Factor VIII proteins having ancestral sequences, expression vectors, and uses related thereto

ABSTRACT

This disclosure relates to recombinant or chimeric FVIII proteins, variants, and vectors encoding the proteins containing one or more ancestral mutations. In certain embodiments, one or more protein domains comprise amino acid sequences that are derived from ancestrally reconstructed amino acid sequences. In certain embodiments, the disclosure relates to pharmaceutical compositions comprising the proteins or vectors and related methods of inducing blood clotting.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/547,742, filed Jul. 31, 2017, which is a U.S. national stage application under 35 USC § 371 of PCTUS2016/015092, filed Jan. 27, 2016, which claims the benefit of U.S. Provisional Application No. 62/109,964 filed Jan. 30, 2015, hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grants R01 HL092179 and U54 HL112309 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is EMY_061US2_SEQ LIST.txt. The text file is 415 KB, was created on May 14, 2020, and is being submitted electronically via EFS-Web. The information the text file is identical to the sequence listing contained in the application.

BACKGROUND

Mutations in the coagulation factor VIII gene result in a decreased or defective coagulation factor (FVIII) protein that gives rise to hemophilia A characterized by uncontrolled bleeding. Treatment of hemophilia A typically entails lifelong, multi-weekly intravenous infusion of either human plasma-derived or recombinant FVIII products. Patients treated with FVIII replacement products often develop neutralizing antibodies that render future treatment ineffective. Thus, there is a need to identify improved therapies.

Gene therapies are typically based on genetically engineered viruses designed to deliver functional transgenes to the patient so that their own cells can biosynthesize a missing or defective protein. Clinical advancements have been made using recombinant adeno-associated viral (rAAV) vectors for the expression of blood factors in the liver. McIntosh et al. report therapeutic levels of FVIII following administration of rAAV vector encoding a human factor VIII variant. Blood. 2013, 121(17):3335-44. See also Brown et al. Molecular Therapy, Methods & Clinical Development (2014) 1, 14036; Doering et al. J. Biol. Chem. 2004, 279:6546-6552; Zakas et al., J Thromb Haemost, 2015, 13(1):72-81; and U.S. Patent Application Publication US20040197875.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to recombinant or chimeric FVIII proteins, variants, and vectors encoding the proteins containing one or more ancestral mutations. In certain embodiments, one or more protein domains comprise amino acid sequences that are derived from ancestrally reconstructed amino acid sequences. In certain embodiments, the disclosure relates to pharmaceutical compositions comprising the proteins or vectors and related methods of inducing blood clotting.

In certain embodiments, ancestral domain mutations are those contained in SEQ ID NO: 2, 11, 20, and 29-38. In certain embodiments, the ancestral domain is a sequence within the A1 domain, A2 domain, activation peptide (ap) domain, A3 domain, C1 domain, or C2 domain optionally having a signaling peptide (sp) domain. In certain embodiments, the FVIII variant comprises a deleted B domain.

In certain embodiments, FVIII variant comprises an A1 domain, an A2 domain, a RHQR sequence (SEQ ID NO: 245), an A3 domain, a C1 domain, and a C2 domain.

In certain embodiments, FVIII variant comprises an A1 domain, an A2 domain, an activation peptide (ap) domain, an A3 domain, a C1 domain, and a C2 domain. In certain embodiments, the FVIII variant optionally comprises a deleted B domain.

In certain embodiments, the FVIII variant comprises a linker of between two and fifty, or two and twenty five, or two and fifteen amino acids between the A2 domain and the an activation peptide (ap) domain.

In certain embodiments, the ancestral sp domain is SEQ ID NO: 3, 12, or 21 or variants thereof. In certain embodiments, the ancestral A1 domain is SEQ ID NO: 4, 13, or 22 or variants thereof.

In certain embodiments, the ancestral A2 domain is SEQ ID NO: 5, 14, or 23 or variants thereof. In certain embodiments, the ancestral B domain is SEQ ID NO: 6, 15, or 24 or variants thereof. In certain embodiments, the ancestral ap domain is SEQ ID NO: 7, 16 or 25 or variants thereof. In certain embodiments, the ancestral A3 domain is SEQ ID NO: 8, 17, or 26 or variants thereof. In certain embodiments, the ancestral C1 domain is SEQ ID NO: 9, 18, or 27 or variants thereof. In certain embodiments, the ancestral C2 domain is SEQ ID NO: 10, 19, or 28 or variants thereof. In certain embodiments, the variants are those having greater than 80, 85, 90, 95, 96, 97, 98, or 99% identity or similarity to the domains. In certain embodiments, the variants are those having one, two, or more amino acid substitutions or conservative substitutions. In certain embodiments, the variants are those having one, two, three, four, five, six seven, eight, nine, or more amino acid substitutions, deletions, or additions.

In certain embodiments, the recombinant FVIII protein has SEQ ID NO: 39, 40, or 42 or variants thereof. In certain embodiments, the variants are those having greater than 80, 85, 90, 95, 96, 97, 98, or 99% identity or similarity to the proteins including or not including the signaling peptide. In certain embodiments, the variants are those having one, two, or more amino acid substitutions or conservative substitutions. In certain embodiments, the variants are those having one, two, three, four, five, six seven, eight, nine, or more amino acid substitutions, deletions, or additions.

In certain embodiments, the disclosure relates to recombinant or chimeric FVIII protein having one or more ancestral sequences or variants thereof such as SEQ ID NOs: 44-244 and 247-277 in the corresponding domains. In certain embodiments, the disclosure relates to recombinant FVIII protein having an ancestral domain selected from a signaling peptide (sp) domain, A1 domain, activation peptide (ap) domain, and A3 domain wherein one or more amino acids in B-domain are optionally deleted or the B-domain has one or more naturally or non-naturally occurring substitutions.

In certain embodiments, the recombinant or chimeric FVIII protein has the E434V mutation as reported in FIG. 1 of US20040197875 wherein the protein has an A2 domain having TDVTF (SEQ ID NO: 43)

In certain embodiments, the disclosure relates to nucleic acids encoding a recombinant or chimeric FVIII protein disclosed herein.

In certain embodiments, the disclosure relates to vectors comprising a promotor nucleic acid sequence in operable combination with a heterologous nucleic acid sequence encoding a recombinant or chimeric FVIII protein disclosed herein. In certain embodiments, the vector comprises a liver-specific promotor and 5′ and 3′ AAV inverted terminal repeats (ITRs).

In certain embodiments, the disclosure contemplates lentiviral gene therapy targeting hematopoietic stem cells. In certain embodiments, the vector is a retroviral or lentiviral vector such as a self-inactivating lentiviral vector derived from HIV-1. In certain embodiments, the vector contains an internal constitutive promoter such as EF1-alpha, PGK or UbC or a cell specific promoter such as GPIb-alpha, CD68, or beta globin LCR.

In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising a recombinant or chimeric FVIII protein and a pharmaceutically acceptable excipient.

In certain embodiments, the disclosure contemplates methods of inducing blood clotting comprising administering an effective amount of a pharmaceutical composition disclosed herein to a subject in need thereof. In certain embodiments, the subject is at risk for, suspected of, exhibiting symptoms of, or diagnosed with a blood clotting disorder, e.g., wherein the subject is diagnosed with hemophilia A or acquired hemophilia.

In certain embodiments, the disclosure contemplates pharmaceutical composition comprising a vector of encoding a protein disclosed herein.

In certain embodiments, the disclosure contemplates method of inducing blood clotting comprising administering an effective amount of a pharmaceutical composition comprising a vector encoding a protein disclosed herein to a subject in need thereof wherein the subject is diagnosed with hemophilia A or acquired hemophilia under conditions such that the protein is expressed inducing blood clotting. In certain embodiments, the subject is unlikely to respond to exogenous FVIII infusions.

In certain embodiments, the disclosure relates to expression systems comprising nucleic acids or vectors comprising nucleic acids disclosed herein.

In certain embodiments, the disclosure relates to methods of inducing blood clotting comprising administering an effective amount of allogenic or autologous cells transduced ex vivo with a vector to express a FVIII disclosed herein.

BRIEF DESCRIPTION OF THE FIGS

FIG. 1 shows a phylogenetic tree that was constructed for mammalian FVIII genes using known amino acid sequences. The ancestral intermediary nodes that were reconstructed through gene synthesis are numbered within the circles. These B-domain deleted genes have been codon optimized for human cell expression and have been sub-cloned into the mammalian expression plasmid ReNeo.

FIG. 2 shows relative FVIII activity data where HEK293T-17 cells were transiently transfected with FVIII plasmid via PEI. Twenty-four hours after replacing DMEM media with serum-free Aim-V, supernatant was assayed for FVIII activity via one-stage coagulation assay and shown as the mean fold increase over human FVIII from two separate experiments. Total activity was normalized to cell counts taken at the time of the FVIII detection and then normalized to human FVIII activity levels.

FIG. 3 shows activity data. Stable BHK-M clones were generated using G418 selection following transfection of FVIII plasmid with Lipofectamine 2000. Twenty-four clones were selected from each FVIII treatment. Shown are the FVIII activity levels of the 4 highest producing clones as determined by one-stage coagulation and normalized to cell counts. Dashed lines indicate the median value for each FVIII treatment. Of the ancestral FVIII sequences, production of nodes 52, 53, and 68 FVIII is significantly different from human FVIII. Furthermore, ancestral nodes 53 and 68 produce significantly higher concentrations of FVIII compared to porcine FVIII.

FIG. 4 shows the decay rate of 1 nM ancestral node 68 FVIIIa determined by chromogenic Xase assay. FVIII protein from the top producing BHK-M clone was collected and purified from serum-free media using one-step ion-exchange chromatography. FVIII was captured with an SPHP HiTrap column and eluted with an increasing concentration of NaCl. Compared to the human FVIII half-life of 1.8 minutes, the ancestral node 68 protein displays a 9-fold prolonged half-life of 16.2 minutes, n=1. Of note, node 68 retains 75% identity to murine FVIII and 71% identity to porcine FVIII.

FIG. 5 shows data on cross-reactivity of inhibitory mouse anti-human monoclonal antibodies (MAbs) to An-53 (black circles) and An-68 (white circles) via direct ELISA. MAbs were selected for high potency (≥1000 BU/mg) and specificity to the A2 or C2 domain. Epitope groups within the domain are listed in parentheses. Cross-reactivity to ancestral FVIII is defined as the percent absorbance compared to human FVIII.

FIG. 6 shows data on the ability to the E434 mutant in A2 to evade 4A4 inhibition.

FIG. 7 shows a schematic of a AAV-FVIII plasmid. FVIII was human codon optimized and subcloned into an AAV expression cassette under the liver-directed HLP promoter.

FIG. 8 shows data comparing An-53 (white) and ET3 (black) FVIII levels following hydrodynamic injection in mice at two doses of AAV-FVIII plasmid DNA. Circles represent 3 μg and downward triangles represent 0.75 μg per mouse. FVIII activity was measured by two-stage chromogenic assay compared to a FACT standard and error bars represent standard deviation of 4 mice.

FIG. 9 shows SDS-PAGE analysis of An-53 and An-68 following two-step ion-exchange. SC: single chain; HC: heavy chain; LC: light chain; cLC: cleaved light chain; A1 and A2: FVIII A domains

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

As used herein, a “non-naturally occurring” sequence is one for which no organisms produce or ever produced through the course of natural events. A protein is non-naturally occurring if one or more amino acids are substituted such that the entire amino acid sequence was never produced due to the course of natural events.

As used herein, an “ancestral mutations” refer to alterations in nucleic acid sequencing that results in amino acid substitutions in the corresponding protein when compared to the consensus human sequence, i.e., SEQ ID NO: 1. A position of the amino acid substitution can be identified by reference to numerical positions within SEQ ID NO: 1 with or without reference to the protein having the signal peptide. With regard to mutations, it is common to refer to the numbering system above wherein FVIII does not contain the signal peptide as it is typically cleaved during cellular processing. As used herein, a “unique ancestral mutation” refers to an ancestral mutation also not found in the sequence for any known primates or mammals.

The terms “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.

As used herein, where “amino acid sequence” is recited herein, it refers to an amino acid sequence of a protein molecule. An “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. However, terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the deduced amino acid sequence, but include post-translational modifications of the deduced amino acid sequences, such as amino acid deletions, additions, and modifications such as glycosylations and addition of lipid moieties.

The term “nucleic acid” refers to a polymer of nucleotides, or polynucleotides. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded of self-complementary, and may include coding regions and regions of various control elements.

The terms “a nucleic acid sequence encoding” a specified polypeptide refers to a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a protein. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide, polynucleotide, or nucleic acid may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present disclosure may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term “recombinant” when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant nucleic acid molecule.

The term “chimera” when used in reference to a polypeptide of polynucleotide refers to the expression product of two or more coding sequences obtained from different genes, that have been cloned together and that, after translation, act as a single polypeptide sequence such that the single or whole polypeptide sequence, or nucleotide sequence, is not naturally occurring. Chimeric polypeptides are also referred to as “hybrid” polypeptides. The coding sequences include those obtained from the same or from different species of organisms.

The term “heterologous nucleic acid” refers to a nucleic acid that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous nucleic acid includes a gene from one species introduced into another species. A heterologous nucleic acid also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous viral nucleic acids are distinguished from endogenous viral genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with viral gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

The terms “variant” and “mutant” when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have “non-conservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).

In certain embodiments, term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

In certain embodiments, sequence “identity” refers to the number of exactly matching amino acids or nucleotide (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. For example the polypeptides GGGGGG and GGGGT have a sequence identity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP and GGGAPPP have a sequence identity of 6 out of 7 or 85%. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic—F Y W; hydrophobic—A V I L; Charged positive: R K H; Charged negative—D E; Polar—S T N Q.

The terms “vector” or “expression vector” refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

Protein “expression systems” refer to in vivo and in vitro and cell free systems. Systems for recombinant protein expression typically utilize cells transfecting with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures. In vitro and cell-free protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vectors, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): e141, both hereby incorporated by reference in their entirety.

In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a polypeptide disclosed herein or chimeric protein thereof.

In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, a beta globin polyA signal, SV40 polyadenylation signal, SV40 origin of replication, Col E1 origin of replication, f1 origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5′ phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil.

Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP-glucose:galactose-1-phosphate uridyltransferaseI (galT), feedback-insensitive a subunit of anthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR), benzyladenine-N-3-glucuronide, E. coli threonine deaminase, glutamate 1-semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSB1), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).

A “label” refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a “label receptor” refers to incorporation of a heterologous polypeptide in the receptor. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

In certain embodiments, the nucleic acids disclosed herein may be a part of any genetic element (vector) which may be delivered to a host cell, e.g., naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc. which transfer the sequences carried thereon. In certain embodiments, a vector may be an adeno-associated virus or human adeno-associated virus (containing AAV genes or sequences) vector, e.g., having nucleic acid sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 serotypes or combinations. In certain embodiments, the nucleic acid sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 serotypes may be at least one or two or more genes or gene fragments of more than 1000, 500, 400, 300, 200, 100, 50, or 25 continuous nucleotides or nucleotides sequences with greater than 50, 60, 70, 80, 90, 95 or 99% identity to the gene or fragment. In certain embodiments, a vector may be a lentivirus based (containing lentiviral genes or sequences) vector, e.g., having nucleic acid sequences derived from VSVG or GP64 pseudotypes or both. In certain embodiments, the nucleic acid sequences derived from VSVG or GP64 pseudotypes may be at least one or two or more genes or gene fragments of more than 1000, 500, 400, 300, 200, 100, 50, or 25 continuous nucleotides or nucleotides sequences with greater than 50, 60, 70, 80, 90, 95 or 99% identity to the gene or fragment.

The selected vector may be delivered by any suitable method, including intravenous injection, ex-vivo transduction, transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. CpG DNA motifs are typically removed because they may lead to gene methylation and silencing. See Bird, DNA methylation and the frequency of CpG in animal DNA, 1980, Nucleic Acids Res, 8: 1499-1504. CpG removal also helps the vector evade immune detection, enhancing the safety and efficacy of the vector. See J Clin Invest. 2013, 123(7):2994-3001, entitled “CpG-depleted adeno-associated virus vectors evade immune detection.”

Blood Coagulation Factors

The blood clothing system is a proteolytic cascade. Blood clotting factors are present in the plasma as a zymogen, in other words in an inactive form, which on activation undergoes proteolytic cleavage to release the active factor from the precursor molecule. The ultimate goal is to produce thrombin. Thrombin converts fibrinogen into fibrin, which forms a clot.

Factor X is the first molecule of the common pathway and is activated by a complex of molecules containing activated factor IX (FIXa), factor VIII, calcium, and phospholipids which are on the platelet surface. Factor VIII is activated by thrombin, and it facilitates the activation of factor X by FIXa. Factor VIII (FVIII), contains multiple domains (A1-A2-B-ap-A3-C1-C2) and circulates in blood in an inactivated form bound to von Willebrand factor (VWF). The C2 domain is involved with FVIII binding to VWF. Thrombin cleaves FVIII causing dissociation with VWF ultimately leading to fibrin formation through factor IX (FIX). Congenital hemophilia A is associated with genetic mutations in the FVIII gene and results in impaired clotting due to lower than normal levels of circulating FVIII. Hemophilia B is similarly associated with genetic mutations in the FIX gene.

A treatment option for a patient diagnosed with hemophilia A is the exogenous administration of recombinant FVIII sometimes referred to as FVIII replacement therapy. In some patients, this therapy can lead to the development of antibodies that bind to the administered FVIII protein. Subsequently, the FVIII-antibody bound conjugates, typically referred to as inhibitors, interfere with or retard the ability of FVIII to cause blood clotting. Inhibitory autoantibodies also sometimes occur spontaneously in a subject that is not genetically at risk of having hemophilia, termed acquired hemophilia. Inhibitory antibodies assays are typically performed prior to exogenous FVIII treatment in order to determine whether the anti-coagulant therapy will be effective.

A “Bethesda assay” has historically been used to quantitate the inhibitory strength the concentration of factor VIII binding antibodies. In the assay, serial dilutions of plasma from a patient, e.g., prior to having surgery, are prepared and each dilution is mixed with an equal volume of normal plasma as a source of FVIII. After incubating for a couple hours, the activities of factor VIII in each of the diluted mixtures are measured. Having antibody inhibitor concentrations that prevent factor VIII clotting activity after multiple repeated dilutions indicates a heightened risk of uncontrolled bleeding. Patients with inhibitor titers after about ten dilutions are felt to be unlikely to respond to exogenous FVIII infusions to stop bleeding. A Bethesda titer is defined as the reciprocal of the dilution that results in 50% inhibition of FVIII activity present in normal human plasma. A Bethesda titer greater than 10 is considered the threshold of response to FVIII replacement therapy.

In certain embodiments, the disclosure relates to methods of inducing blood clotting comprising administering an effective amount of a recombinant or chimeric protein disclosed herein or viral particle or capsid comprising a vector comprising a nucleic acid encoding a blood clotting factor as disclosed herein to a subject in need thereof.

In certain embodiments, the subject is diagnosed with hemophilia A or acquired hemophilia or unlikely to respond to exogenous FVIII infusions.

In certain embodiments, the protein, capsid, or vector is administered in combination with an immunosuppressive agent, e.g., ciclosporin, tacrolimus, sirolimus, cyclophosphamide, methotrexate, azathioprine, mercaptopurine, fluorouracil, mycophenolic acid, dactinomycin, fingolimod, T-cell receptor antibody or binding protein, muromonab-CD3, IL-2 receptor antibody or binding protein, basiliximab, daclizumab, recombinant IFN-beta, TNF-alpha antibody or binding protein, infliximab, etanercept, adalimumab, or combinations thereof.

In certain embodiments, modified cells are re-administered in combination with irradiation, busulfan, and/or anti-thymocyte globulin, e.g., when using lentiviral gene therapy targeting hematopoietic stem cells.

Treating patients with inhibitors to FVIII has also been accomplished by methods of immune tolerance induction (ITT) which typically involves the daily infusion of FVIII until circulating inhibitor/antibody levels decline. However, 20-30% of patients fail to become tolerant after an immune tolerance induction (ITT) therapy. Persistence of FVIII inhibitors is associated with increased risks of morbidity and mortality. In certain embodiments, the disclosure relates to methods of immune tolerance induction comprising administering an effective amount of a recombinant or chimeric protein disclosed herein or a vector or a capsid as disclosed herein to a subject in need thereof.

Vector-Mediated Gene Transfer

In certain embodiments, the disclosure relates to methods of treating a subject diagnosed with a genetic trait that results in expression of a mutated or truncated non-functional protein by administering an effective amount of a vector disclosed herein. In certain embodiments, the vector is configured to express a functional protein from the liver. In certain embodiments, the vector is configured to express a protein in any cells such as hematopoietic stem cells or other hematocytes in vivo or by ex vivo transduction.

As used herein, the liver specific promotor refers to the sequences in the 5′ direction of the transcriptional start site of the protein to be produced. McIntosh et al. report an rAAV vector encoding a human factor VIII variant using a hybrid liver-specific promoter (HLP). Blood. 2013, 121(17):3335-44. Brown et al. report liver-directed adeno-associated viral vector delivery of a bioengineered FVIII. Molecular Therapy, Methods & Clinical Development (2014) 1, 14036.

In certain embodiments, the vector comprises a viral nucleic acid sequence of greater than 10, 20, 30, 40, 50, 100, or 200 nucleotides. In certain embodiments, the viral nucleic acid sequence is a segment of human adeno-associated virus (hAAV) of serotypes 1, 2, 3B, 4, 5, 6, 7, 8, 9 or combinations or variants thereof, typically comprising an AAV inverted terminal repeat.

Adeno-associated virus (AAV), Parvovirus family, is an icosahedral virus with single-stranded linear DNA genomes. The life cycle of AAV includes a latent phase at which AAV genomes, after infection, are site specifically integrated into host chromosomes and an infectious phase in which, following either adenovirus or herpes simplex virus infection, the integrated genomes are subsequently rescued, replicated, and packaged into infectious viruses. The properties of non-pathogenicity, broad host range of infectivity, including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.

The nucleic acid and promotor sequences disclosed herein are useful in production of rAAV, and are also useful as antisense delivery vectors, gene therapy vectors, or vaccine vectors. In certain embodiments, the disclosure provides for gene delivery vectors, and host cells which contain the nucleic acid sequences disclosed herein.

In certain embodiments, the disclosure relates to virus particles, e.g., capsids, containing the nucleic acid sequences encoding promotors and proteins disclosed herein. The virus particles, capsids, and recombinant vectors are useful in delivery of a heterologous gene or other nucleic acid sequences to a target cell. The nucleic acids may be readily utilized in a variety of vector systems, capsids, and host cells. In certain embodiments, the nucleic acids are in vectors contained within a capsid comprising cap proteins, including AAV capsid proteins vp1, vp2, vp3 and hypervariable regions.

Adeno-associated virus (AAV) has a biphasic life cycle consisting of both a productive and a latent phase. In the presence of helper virus, Adenovirus (Ad), or Herpesvirus (HSV), AAV undergoes a productive infection. Lacking a helper virus, AAV latently infects by integration into the host genome. AAV is capable of undergoing site-specific integration into the human genome. The ability to integrate site specifically is one of the attractive features for using this virus as a vector for human gene therapy.

Inverted terminal repeats (ITRs) in AAVs are cis elements used for targeted integration. Traditional AAV-mediated site-specific integration uses the AAV Rep78/68 proteins and ITRs which contain the Rep-binding site (RBS) and AAVS1 which is a sequence present at the integration site. See Linden et al. (1996) entitled “The recombination signals for adeno-associated virus site-specific integration.” Proc Natl Acad Sci USA 93(15):7966-7972.

AAV vectors typically contain a transgene expression cassette between the ITRs that replaces the rep and cap genes. Vector particles are produced by the co-transfection of cells with a plasmid containing the vector genome and a packaging/helper construct that expresses the rep and cap proteins in trans. During infection, AAV vector genomes enter the cell nucleus and can persist in multiple molecular states. One common outcome is the conversion of the AAV genome to a double-stranded circular episome by second-strand synthesis or complementary strand pairing.

The AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and function variants thereof. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

In certain embodiments, this disclosure provides for a nucleic acids disclosed herein the encoded proteins or wherein the nucleic acids are part of an expression cassette or transgene. See e.g., US Pat. App. Pub. 20150139953. In certain embodiments, the expression cassette is composed of a transgene and regulatory sequences, e.g., promotor and 5′ and 3′ AAV inverted terminal repeats (ITRs). In one desirable embodiment, the ITRs of AAV serotype 2 or 8 are used. However, ITRs from other suitable serotypes may be selected. An expression cassette is typically packaged into a capsid protein and delivered to a selected host cell.

In certain embodiments, the disclosure provides for a method of generating a recombinant adeno-associated virus (AAV) having an AAV serotype capsid, or a portion thereof. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype capsid protein; a functional rep gene; an expression cassette composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. See e.g., US Pat. App. Pub. 20150139953.

The components for culturing in the host cell to package an AAV expression cassette in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the components (e.g., expression cassette, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

Introduction into the host cell of the vector may be achieved by any means known in the art or as disclosed above, including transfection, infection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion, among others. One or more of the adenoviral genes may be stably integrated into the genome of the host cell, stably expressed as episomes, or expressed transiently. The gene products may all be expressed transiently, on an episome or stably integrated, or some of the gene products may be expressed stably while others are expressed transiently. Furthermore, the promoters for each of the adenoviral genes may be selected independently from a constitutive promoter, an inducible promoter or a native adenoviral promoter. The promoters may be regulated by a specific physiological state of the organism or cell (i.e., by the differentiation state or in replicating or quiescent cells) or by exogenously added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the host cell may be accomplished using techniques known to the skilled artisan. In preferred embodiment, standard transfection techniques are used, e.g., CaPO₄ transfection or electroporation, and/or infection by hybrid adenovirus/AAV vectors into cell lines such as the human embryonic kidney cell line HEK 293 (a human kidney cell line containing functional adenovirus E1 genes which provides trans-acting E1 proteins).

One of skill in the art will readily understand that the AAV techniques can be adapted for use in these and other viral vector systems for in vitro, ex vivo or in vivo gene delivery. The in certain embodiments the disclosure contemplates the use of nucleic acids and vectors disclosed herein in a variety of rAAV and non-rAAV vector systems. Such vectors systems may include, e.g., lentiviruses, retroviruses, poxviruses, vaccinia viruses, and adenoviral systems, among others.

For example, in certain embodiments, the disclosure contemplates expression of FVIII by lentiviral vectors through in vivo administration or ex vivo transduction. Allogenic or autologous CD34+ cells can be isolated and mixed ex vivo with a lentiviral vector having nucleic acids encoding a FVIII protein disclosed herein under conditions such that the lentiviral vector integrates into the cells nucleic acids, e.g. DNA, sufficiently to produce or allow production the encoded FVIII protein. The cells are then infused or re-infused into a subject, wherein the subject optionally received a myeloablative treatment, under conditions such that hematopoietic stem cells or resulting cells or hematocytes express the protein. See Cartier et al. Science. 2009, 326(5954):818-23. Sanber et al. report the construction of stable packaging cell lines for clinical lentiviral vector production. Sci Rep. 2015, 5:9021. Hu et al. report production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors. See Mol Ther Methods Clin Dev. 2015, 2:15004.

Therapeutics

Recombinant or chimeric proteins and virus particles, capsids, or vectors encoding proteins disclosed herein can be delivered, e.g., to liver via the hepatic artery, the portal vein, or intravenously to yield therapeutic levels of therapeutic proteins or clotting factors in the blood. The recombinant or chimeric proteins and capsid or vector is preferably suspended in a physiologically compatible carrier, may be administered to a patient. Suitable carriers may be readily selected by one of skill in the art in view of the indication. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, sesame oil, and water.

Optionally, the compositions of the disclosure may contain other pharmaceutically acceptable excipients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The recombinant virus particles, capsids, or vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., the liver (optionally via the hepatic artery) or lung), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

Dosages of the recombinant or chimeric proteins and virus particles, capsids, or vectors will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of the protein is generally in the range of from about 0.1 ml to about 100 ml of solution containing concentrations of from about 1×10⁹ to 1×10¹⁶ genomes, virus vectors, or gene modified cells produced by ex vivo transduction.

Therapeutic proteins encoded by the nucleic acids (e.g., operably in combination with promoters) reported herein include those used for treatment of hemophilia, including hemophilia hemophilia A (including Factor VIII and its variants, such as the light chain and heavy chain of the heterodimer and the B-deleted domain; U.S. Pat. Nos. 6,200,560 and 6,221,349). The Factor VIII gene codes for 2351 amino acids and the protein has six domains, designated from the amino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al, Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Toole et al, Nature, 342:337 (1984)]. Human Factor VIII is processed within the cell to yield a heterodimer primarily comprising a heavy chain containing the A1, A2 and B domains and a light chain containing the A3, C1 and C2 domains. Both the single chain polypeptide and the heterodimer circulate in the plasma as inactive precursors, until activated by thrombin cleavage between the A2 and B domains, which releases the B domain and results in a heavy chain consisting of the A1 and A2 domains. The B domain is deleted in the activated procoagulant form of the protein. Additionally, in the native protein, two polypeptide chains (“a” and “b”), flanking the B domain, are bound to a divalent calcium cation.

Administration of only the chain defective is contemplated in patients because most persons with hemophilia contain a mutation or deletion in only one of the chains (e.g., heavy or light chain). Thus, in certain embodiments, the disclosure relates to recombinant or chimeric proteins of a light chain containing the A3, C1 and C2 domains or a heavy chain consisting of the A1 and A2 domains. In some embodiments, the therapeutic protein or as encoded by the nucleic acids (e.g., operably in combination with promoters) reported herein comprises the Factor VIII heavy chain which encodes the 19 amino acid signal sequence, as well as the human beta globin polyadenylation sequence or growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the A1 and A2 domains, as well as 5 amino acids from the N-terminus of the B domain, and/or 8 amino acids of the C-terminus of the B domain, as well as the A3, C1 and C2 domains. In yet other embodiments, the protein or nucleic acids encoding Factor VIII is a heavy chain and light chain provided in a single protein or nucleic acid separated by 42 nucleic acids/14 amino acids of the B domain. See U.S. Pat. No. 6,200,560.

As used herein, a therapeutically effective amount is an amount of protein or vector that produces sufficient amounts of Factor VIII to decrease the time it takes for the blood of a subject to clot. Generally, severe hemophiliacs having less than 1% of normal levels of Factor VIII have a whole blood clotting time of greater than 60 minutes as compared to approximately 10 minutes for non-hemophiliacs.

The recombinant or chimeric proteins or vectors may contain an amino acid sequence or nucleic acid coding for fragments of Factor VIII which is itself not biologically active, yet when administered into the subject improves or restores the blood clotting time. For example, the Factor VIII protein comprises two polypeptide chains: a heavy chain and a light chain separated by a B-domain which is cleaved during processing. Co-transducing recipient cells with the Factor VIII heavy and light chains leads to the expression of biologically active Factor VIII. Administration of only the chain defective is contemplated in patients because most hemophiliacs contain a mutation or deletion in only one of the chains (e.g., heavy or light chain).

Thus, in certain embodiments, the disclosure relates to vectors disclosed herein having nucleic acids encoding a light chain containing the A3, C1 and C2 domains or a heavy chain consisting of the A1 and A2 domains.

Bioengineering Coagulation Factor VIII Through Ancestral Protein Reconstruction

The development of transformative hemophilia A therapeutics has been hindered by the size, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of FVIII orthologs from existing vertebrate species, unique molecular, cellular and biochemical properties that can overcome the limitations of human FVIII were discovered. This approach facilitated the development of recombinant porcine FVIII for acquired hemophilia A and has enabled low resolution mapping and bioengineering of functional sequence determinants into human FVIII.

A mammalian FVIII phylogenetic tree with corresponding ancestral node (An) sequences was constructed through Bayesian inference using both DNA and amino acid-based models in PAML Version 4.1 (FIG. 1). Initially, 14 An-FVIII sequences were selected for reconstruction and subsequent molecular, cellular, biochemical and immunological characterization. Each An-FVIII displayed activity in coagulation assays utilizing human hemophilia A plasma as a substrate thus demonstrating evolutionary mammalian compatibility. Infusion of highly purified preparations of several An-FVIIIs into hemophilia A mice also corrected the bleeding phenotype following a tail transfection bleeding challenge confirming in vivo functionality.

To study biosynthetic efficiency, secreted FVIII activity and mRNA transcript levels were analyzed following transfection of An-FVIII plasmids into HEK293 and BHK-M cell lines. An-53, common ancestor to rodents and primates, and An-68, ancestor to a subset of current rodents, displayed the highest FVIII biosynthetic efficiencies that were 12 and 15 fold greater than human FVIII, respectively. These two An-FVIII sequences share 95 and 87% amino acid identity to human FVIII, respectively. In contrast, intermediate ancestors between An-53 and human FVIII, designated An-55, -56 and -57, do not display enhanced biosynthetic efficiency suggesting that the functional sequence determinant of high expression was lost during primate evolution.

Testing whether high expression ancestral FVIIIs would be enabling to gene therapy approaches, An-53, An-68 and human FVIII cDNAs were placed in an AAV expression cassette under the control of a liver-directed promoter and the resulting plasmid DNA was infused hydrodynamically into hemophilia A mice. An-53 and An-68, but not human FVIII vector treated animals, achieved sustained, therapeutic plasma FVIII activity levels over 4 weeks (0.1-0.6 IU/ml versus <0.01 IU/ml, respectively). Recombinant An-FVIIIs were expressed, purified and biochemically characterized by SDS-PAGE, specific activity, decay following thrombin activation and inhibitor recognition. Early mammalian and all primate lineage thrombin-activated An-FVIII(a) displayed half-lives between 1.5-2.2 min that were not distinguishable from human FVIII.

Modern murine, porcine, and ovine FVIIIa display significantly longer half-lives. An-68 and An-78 display prolonged half-lives of 16 and 7 min, respectively. Lastly, the immune recognition of An-FVIIIs by a panel of A2 and C2 domain targeting inhibitory murine monoclonal antibodies as well as hemophilia A inhibitor patient plasmas was examined and many examples of reduced reactivity were revealed, which may enable the development of less immunogenic FVIII products.

Factor VIII domain boundaries refer to the human FVIII amino acid sequence numbering as follows; residues 1-19 (Signal Sequence), 20-391 (A1), 392-759 (A2), 760-1667 (B), 1668-1708 (ap), 1709-2038 (A3), 2039-2191 (C1) and 2192-2351 (C2). Gitschier et al., Nature, 1984, 312, 326-330. (SEQ ID NO: 1):

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP PRVPKSFPFN 61 TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA 421 PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNSRHPST RQKQFNATTI 781 PENDIEKTDP WFAHRTPMPK IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS PGAIDSNNSL SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSST SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSTESGR LFKGKRAHGP ALLTKDNALF KVSISLLKTN 1021 KTSNNSATNR KTHIDGPSLL IENSPSVWQN ILESDTEFKK VTPLIHDRML MDKNATALRL NHMSNKTTSS KNMEMVQQKK EGPIPPDAQN PDMSFFKMLF LPESARWIQR THGKNSLNSG QGPSPKQLVS LGPEKSVEGQ NFLSEKNKVV VGKGEFTKDV GLKEMVFPSS RNLFLTNLDN 1201 LHENNTHNQE KKIQEEIEKK ETLIQENVVL PQIHTVTGTK NFMKNLFLLS TRQNVEGSYD GAYAPVLQDF RSLNDSTNRT KKHTAHFSKK GEEENLEGLG NQTKQIVEKY ACTTRISPNT SQQNFVTQRS KRALKQFRLP LEETELEKRI IVDDTSTQWS KNMKHLTPST LTQIDYNEKE 1381 KGAITQSPLS DCLTRSHSIP QANRSPLPIA KVSSFPSIRP IYLTRVLFQD NSSHLPAASY RKKDSGVQES SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT YKKVENTVLP 1501 KPDLPKTSGK VELLPKVHIY QKDLFPTETS NGSPGHLDLV EGSLLQGTEG AIKWNEANRP GKVPFLRVAT ESSAKTPSKL LDPLAWDNHY GTQIPKEEWK SQEKSPEKTA FKKKDTILSL 1621 NACESNHAIA AINEGQNKPE IEVTWAKQGR TERLCSQNPP VLKRHQREIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR 1741 AQSGSVPQFK KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD CKAWAYFSDV 1861 DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD SSGIKHNIFN 2161 PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM 2341 EVLGCEAQDL Y

HSQ is B domain deleted human factor VIII. ET3 is a B domain deleted FVIII hybrid that contains human and porcine domains, i.e., sequence (A1 and A3 porcine) with a linker in the deleted B domain. ET3 utilizes a 24 amino acid porcine sequence-derived OL linker sequence, i.e., porcine-derived sequence SFAQNSRPPSASAPKPPVLRRHQR (SEQ ID NO: 246). Both HSQ and ET3 contain the RHQR (SEQ ID NO: 245) recognition sequence for PACE/furin processing sequence for the B-domain.

In certain embodiments, the recombinant or chimeric FVIII protein further comprises a linker amino acid sequence between two and fifty, or two and twenty five, or two and fifteen amino acids between the A2 domain and the an activation peptide (ap) domain. In certain embodiments, the linker comprises RHQR (SEQ ID NO: 245), SRPPSASAPK (SEQ ID NO: 41), or SFAQNSRPPSASAPKPPVLRRHQR (SEQ ID NO: 246).

In certain embodiments, the disclosure relates to FVIII proteins comprising one, two, more, or combinations of ancestral mutation(s). In certain embodiments, the disclosure relates to a recombinant or chimeric FVIII protein comprising one or more ancestral mutations wherein optionally one or more amino acids in B-domain are deleted, and wherein the sequence does not naturally occur.

The sequence for Node number 68 is: SEQ ID NO: 2

MQIALFTCFFLSLFNFCSSATRRYYLGAVELSWNYMQSDLLSVLHTDTRFL PRMPTSFPFNTSIMYKKTVFVEYMDHLFNIAKPRPPWMGLLGPTIWTEVHD TVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVFPGES HTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCKEGSL SKERTQMLHQFVLLFAVFDEGKSWHSETKDSFTQAMDSASTRAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASL EISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCPEEPQWQK KNNEEMEDYDDDLDSEMDMFTLDDDNSPFIQIRSVAKKYPKTWIHYISAEE EDWDYAPSVLTSDDGSYKSQYLSNGPHRIGRKYKKVRFIAYTDETFKTRET IQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSPLHSRRLP RGIKHVKDLPIRPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDL ASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENQSWYITENMQRF LPNAADTQPQDPEFQASNIMHSINGYVFDSLQLTVCLHEVAYWYILSVGAQ TDFLSIFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSD FRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQLVNDNNVIEPRSFFQNSN HPNTRKKKFKATTIPENDIEKIEPQFGETAEMLKVQSVSSSDLLMLLGQSP TPHGLSLSDNQEAIYEAIPDDHSPDAIDSNEGPSKVTQLRPELHHSGKIVF TPEPGLQLRSNKNLETTIEVKWKKLDLQVSSLPNNLMTTTILSDNLTATSE KTDSSGSPDMPVHFSSKLSTTAFGKKSYPLIGSHVPLNISERNSDSNLLDA TLMNNQESSLGDNISSMENDRLLKEKRFHGIALLTKDNTLFKDNISLMKTN KTYNHSTTNGKAHIDSPTSIENSTAVLQDTILKINSEIQEVTSLIHDGTLS GKNTTYLRLNHMLNRTTSSKNKEIFHQKDEDPVPQDTENTIMPFFKMLFLP ESANWMKRTNGNNSLNSEQGPSPKQLVYLMLEKSVKNQNFLSEKNKVIVEQ DEFTKDTGLKDMVFPSNMSIFLTTLANVQENDMHNQEKNIQEEIEKEALIE EKVVLPQVHIATGSKNFLKDIFFLGTRQNVSLDEDIYVPVLQDISSINNST NTVEIHMAHFFKRREDEETNSEGLVNKTREMVKNYPSQKNIITQRSKRALG QFRLPLASTQWPQTMNYLTQSIITQIDHSKEGEKSITQSSLSDSSMIKSTT QTNSSGLHIVKTSAFPPTDLKRIPFQDKFFHVLASSYTYDFKTKSSRIQES SHFLKETKINNSSLAILPWEMIINQGKFASPGTSNTNSVTYKKLENIVLLK PVLPEESGKVELLPQVSIHEEELLPTETSHGSPGHLDLMKEVFLQKTQGPI KWNKAKRHGESELKGTTESSEKTPSKLLDHLAWDNHYAAQIPKDKWKSKEK SPEITSIKREDTILSLNPHENNHSIVANEKQNWPQREATWVKQGQTQRLCS QNPPVLKRHQRELSALQSEQEATDYDDSMETNEDFDIYGEDIKQGPRSFQQ KTRHYFIAAVERLWDYGMSTSPHVLRNRDQSGNAPQFKKVVFQEFTDGSFS QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKED QRQGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLERD MHSGLIGPLLICHTNTLNPAHGRQVAVQEFALFFTIFDETKSWYFTENVER NCKTPCNIQMEDPTLKENYRFHAINGYVMDTLPGLVMAQDQRIRWYLLSMG SNENIQSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSRAGIWRVE CLIGEHLQAGMSTLFLVYSKQCQIPLGMASGSIRDFQITASGHYGQWAPNL ARLHHSGSINAWSTKEPFSWIKVDLLTPMIIHGIKTQGARQKFSSLYISQF IIMYSLDGKKWLSYRGNSTGTLMVFFGNVDSSGIKHNSFNPPIIARYIRLH PTHSSIRSTLRMELMGCDLNSCSIPLGMENKVISDTQITASSYFTNMFATW SPSQARLHLQGRTNAWRPQVNDPKEWLQVDLQKTMKVTGIITQGVKSLFTS MFVKEFLISSSQDGHHWTHILHNGKVKVFQGNQDSSTPMVNSLDPPLLTRY LRIHPQIWEHQIALRLEILGCEAQQLY Accordingly the sequences for sp-A1-A2-B-aq-A3-C1-C2:

(signal peptide, sp domain, SEQ ID NO: 3) MQIALFTCFFLSLFNFCS S (A1 domain SEQ ID NO: 4) ATRRYYLGAVELSWNYMQSDLLSVLHTDTRFLPRMPTSFPFNTSIMYKKTV FVEYMDHLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYEDQTSQREKEDDKVFPGESHTYVWQVLKENGPMASDPP CLTYSYLSHVDLVKDLNSGLIGALLVCKEGSLSKERTQMLHQFVLLFAVFD EGKSWHSETKDSFTQAMDSASTRAWPKMHTVNGYVNRSLPGLIGCHRKSVY WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLIDLGQ FLLFCHISSHKHDGMEAYVKVDSCPEEPQWQKKNNEEMEDYDDDLDSEMDM FTLDDDNSPFIQIRS (A2 domain SEQ ID NO: 5) VAKKYPKTWIHYISAEEEDWDYAPSVLTSDDGSYKSQYLSNGPHRIGRKYK KVRFIAYTDETFKTRETIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNI YPHGITDVSPLHSRRLPRGIKHVKDLPIRPGEIFKYKWTVTVEDGPTKSDP RCLTRYYSSFINPERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFS VFDENQSWYITENMQRFLPNAADTQPQDPEFQASNIMHSINGYVFDSLQLT VCLHEVAYWYILSVGAQTDFLSIFFSGYTFKHKMVYEDTLTLFPFSGETVF MSMENPGLWVLGCHNSDFRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQL VNDNNVIEPR (B domain SEQ ID NO: 6) SFFQNSNHPNTRKKKFKATTIPENDIEKIEPQFGETAEMLKVQSVSSSDLL MLLGQSPTPHGLSLSDNQEAIYEAIPDDHSPDAIDSNEGPSKVTQLRPELH HSGKIVFTPEPGLQLRSNKNLETTIEVKWKKLDLQVSSLPNNLMTTTILSD NLTATSEKTDSSGSPDMPVHFSSKLSTTAFGKKSYPLIGSHVPLNISERNS DSNLLDATLMNNQESSLGDNISSMENDRLLKEKRFHGIALLTKDNTLFKDN ISLMKTNKTYNHSTTNGKAHIDSPTSIENSTAVLQDTILKINSEIQEVTSL IHDGTLSGKNTTYLRLNHMLNRTTSSKNKEIFHQKDEDPVPQDTENTIMPF FKMLFLPESANWMKRTNGNNSLNSEQGPSPKQLVYLMLEKSVKNQNFLSEK NKVIVEQDEFTKDTGLKDMVFPSNMSIFLTTLANVQENDMHNQEKNIQEEI EKEALIEEKVVLPQVHIATGSKNFLKDIFFLGTRQNVSLDEDIYVPVLQDI SSINNSTNTVEIHMAHFFKRREDEETNSEGLVNKTREMVKNYPSQKNIITQ RSKRALGQFRLPLASTQWPQTMNYLTQSIITQIDHSKEGEKSITQSSLSDS SMIKSTTQTNSSGLHIVKTSAFPPTDLKRIPFQDKFFHVLASSYTYDFKTK SSRIQESSHFLKETKINNSSLAILPWEMIINQGKFASPGTSNTNSVTYKKL ENIVLLKPVLPEESGKVELLPQVSIHEEELLPTETSHGSPGHLDLMKEVFL QKTQGPIKWNKAKRHGESELKGTTESSEKTPSKLLDHLAWDNHYAAQIPKD KWKSKEKSPEITSIKREDTILSLNPHENNHSIVANEKQNWPQREATWVKQG QTQRLCSQNPPVLKRHQR (ap domain SEQ ID NO: 7) ELSALQSEQEATDYDDSITIETNEDFDIYGEDIKQGPR (A3 domain SEQ ID NO: 8) SFQQKTRHYFIAAVERLWDYGMSTSPHVLRNRDQSGNAPQFKKVVFQEFTD GSFSQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLIS YKEDQRQGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAYFSDVD LERDMHSGLIGPLLICHTNTLNPAHGRQVAVQEFALFFTIFDETKSWYFTE NVERNCKTPCNIQMEDPTLKENYRFHAINGYVMDTLPGLVMAQDQRIRWYL LSMGSNENIQSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSRAGI WRVECLIGEHLQAGMSTLFLVYSK (C1 domain SEQ ID NO: 9) QCQIPLGMASGSIRDFQITASGHYGQWAPNLARLHHSGSINAWSTKEPFSW IKVDLLTPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWLSYRGNSTG TLMVFFGNVDSSGIKHNSFNPPIIARYIRLHPTHSSIRSTLRMELMGCDLN (C2 domain SEQ ID NO: 10) SCSIPLGMENKVISDTQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQV NDPKEWLQVDLQKTMKVTGIITQGVKSLFTSMFVKEFLISSSQDGHHWTHI LHNGKVKVFQGNQDSSTPMVNSLDPPLLTRYLRIHPQIWEHQIALRLEILG CEAQQLY The sequence for Node number 54 is: SEQ ID NO: 11

MQIELSTCFFLCLLQFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRFP PRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYD TVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGES HTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AKERTQTLHKFVLLFAVFDEGKSWHSETKDSLMQDMDSASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASL EISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRM KNNEEEEDYDDDLADSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAA EEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTR EAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLHSGR LPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINLER DLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSWYLTENMQ RFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIG AQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHN SDFRNRGMTALLKVSSCDRNTGDYYEDTYEDISTYLLSENNVIEPRSFSQN SRHPSTRQKQFKATTIPENDIEKIDPQFGERTQMLKVQSVSSSDLLMLLGQ SPTPHGLSLSDLQEATYEAIPDDHSPGAIDSNEGPSEVAHLRPELHHSGDM VFTPEPGLQLRLNENLGTTIAVELKKLDLKVSSSSNNLMTSPTIPSDNLAA GTEKTGSLGPPNMPVHFDSQLDTTVFGKKSSPLIGSGVPLSLSERNNDSKL LEAALMNSQESSLGKNVSSMESDRLFKEKRAHGPALLTKDNALFKVNISLV KTNKTSNNSTTNRKTHIDGPTLLIENSTSVWQDILESDTEFQEVTSLIHDK MLMDKNTTALRLNHVSNKTTSSKNMEMVHQKKEGPVPPDAENPDMSFFKML FLPESANWIKRTHGKNSLNSGQGPSPKQLISLGSEKSVKDQNFLSEKNKVV VGEDEFTKDTGLKEMIFPSSRNIFLTNLANVHENDTHNQEKKIQEEIERKE TLIQENVVLPQVYTVTGTKNFMKNLFLLSTRQNVEGLDEGAYAPVLQDTRS LNDSTNRTEIHMAHFSKKREEENLEGLGNQTKQMVEKYPSTTRMSPNPSQQ NVITQRGKRALKQFRLPLEETELEKGLIVDDTSTQWSKNMKYLTQSTLTQI DYNEKEKKAITQSPLSDCLMRNHSITQTNSSALPIAKVSAFPSIRPTDLTR VPSQDNSSHLLASAYRKKSSGVQESSHFLQGAKRNNLSLAILTLEMIGNQR KVGSLGTSATNSVMYKKLENTVLLKPGLPEASGKVELLPKVHIHQKDLFPT ETSNGSPGHLDLMEEILLQKTQGAIKWNKTNRPGKVPFLKGATESSEKTPS KLLGPLAWDNQYATQIPKEEWKSQEKSPENTAFKTKDTILSLNPCESNHAI AAINEGQDRPQREATWAKQGGTGRLCSQNPPVLKRHQREITLTTLQSDQEE IDYDDTISIETKREDFDIYGEDENQSPRSFQKKTRHYFIAAVERLWDYGMS RSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFKNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKT YFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICHTNTLNP AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENY RFHAINGYVMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK KEEYKMAVYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYS KQCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNST GTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDL NSCSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQ VNNPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTL FFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLEVL GCEAQQLY Accordingly the sequences for sp-A1-A2-B-aq-A3-C1-C2:

(sp, Signal Peptide, SEQ ID NO: 12) MQIELSTCFFLCLLQFSFS (A1 SEQ ID NO: 13) ATRRYYLGAVELSWDYMQSDLLSELHVDTRFPPRVPRSFPFNTSVMYKKTV FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAV GVSYWKASEGAEYDDQTSQREKEDDKVFPGESHTYVWQVLKENGPMASDPP CLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKERTQTLHKFVLLFAVFD EGKSWHSETKDSLMQDMDSASARAWPKMHTVNGYVNRSLPGLIGCHRKSVY WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQ FLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEEEDYDDDLADSEMD VVRFDDDNSPSFIQIRS (A2 SEQ ID NO: 14) VAKKHPKTWVHYIAAEEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKYK KVRFMAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNI YPHGITDVRPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDP RCLTRYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFS VFDENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLS VCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF MSMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDISTYL LSENNVIEPR (B SEQ ID NO: 15) SFSQNSRHPSTRQKQFKATTIPENDIEKIDPQFGERTQMLKVQSVSSSDLL MLLGQSPTPHGLSLSDLQEATYEAIPDDHSPGAIDSNEGPSEVAHLRPELH HSGDMVFTPEPGLQLRLNENLGTTIAVELKKLDLKVSSSSNNLMTSPTIPS DNLAAGTEKTGSLGPPNMPVHFDSQLDTTVFGKKSSPLIGSGVPLSLSERN NDSKLLEAALMNSQESSLGKNVSSMESDRLFKEKRAHGPALLTKDNALFKV NISLVKTNKTSNNSTTNRKTHIDGPTLLIENSTSVWQDILESDTEFQEVTS LIHDKMLMDKNTTALRLNHVSNKTTSSKNMEMVHQKKEGPVPPDAENPDMS FFKMLFLPESANWIKRTHGKNSLNSGQGPSPKQLISLGSEKSVKDQNFLSE KNKVVVGEDEFTKDTGLKEMIFPSSRNIFLTNLANVHENDTHNQEKKIQEE IERKETLIQENVVLPQVYTVTGTKNFMKNLFLLSTRQNVEGLDEGAYAPVL QDTRSLNDSTNRTEIHMAHFSKKREEENLEGLGNQTKQMVEKYPSTTRMSP NPSQQNVITQRGKRALKQFRLPLEETELEKGLIVDDTSTQWSKNMKYLTQS TLTQIDYNEKEKKAITQSPLSDCLMRNHSITQTNSSALPIAKVSAFPSIRP TDLTRVPSQDNSSHLLASAYRKKSSGVQESSHFLQGAKRNNLSLAILTLEM IGNQRKVGSLGTSATNSVMYKKLENTVLLKPGLPEASGKVELLPKVHIHQK DLFPTETSNGSPGHLDLMEEILLQKTQGAIKWNKTNRPGKVPFLKGATESS EKTPSKLLGPLAWDNQYATQIPKEEWKSQEKSPENTAFKTKDTILSLNPCE SNHAIAAINEGQDRPQREATWAKQGGTGRLCSQNPPVLKRHQR (ap SEQ ID NO: 16) EITLTTLQSDQEEIDYDDTISIETKREDFDIYGEDENQSPR (A3 SEQ ID NO: 17) SFQKKTRHYFIAAVERLWDYGMSRSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLIS YEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDMHSGLIGPLLICHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTE NMERNCRAPCNIQMEDPTFKENYRFHAINGYVMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSKAGI WRVECLIGEHLHAGMSTLFLVYSK (C1 SEQ ID NO: 18) QCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTG TLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLN (C2 SEQ ID NO: 19) SCSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQV NNPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTLF FQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLEVLG CEAQQLY The sequence for Node number 53 is: SEQ ID NO: 20

MQIELSTCFFLCLLQFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHQFVLLFAVFDEGKSWHSETKDSLTQAMDSASARAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSEMDVVRFDDDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTYL LSENNVIEPRSFSQNSRHPSTRQKQFKATTIPENDIEKIDPQFGERTQML KVQSVSSSDLLMLLGQSPTPHGLSLSDLQEATYEAIPDDHSPGAIDSNEG PSEVAHLRPELHHSGDMVFTPEPGLQLRLNENLGTTIAVELKKLDLKVSS SSNNLMTSPTIPSDNLAAGTEKTGSLGPPNMPVHFSSQLGTTVFGKKSSP LIGSGVPLNLSERNNDSKLLEAALMNSQESSLGKNVSSMESDRLFKEKRV HGPALLTKDNALFKVNISLVKTNKTSNNSTTNRKTHIDGPTLLIENSTSV WQDIILESDTEFQEVTSLIHDEMLMDKNTTALRLNHVSNKTTSSKNMEMV HQKKEGPVPPDAENPDMSFFKMLFLPESANWIKRTHGKNSLNSGQGPSPK QLISLGSEKSVKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNSRSIFLT NLANVQENDTHNQEKKFQEEIERKETLIQENVVLPQVYTVTGTKNFLKNL FLLSTRQNVEGLDEGTYAPVLQDTRSLNDSTNRAEIHMAHFSKRREEENL EGLGNQTKQMVEKYPSTTRMSPNPSQQNVITQRGKRALKQFRLPLEETEL EKGLIVDDTSTQWSKNMKYLTQSTLTQIDYNEKEKKAITQSPLSDCSMRN HSITQTNSSALPIAKVSAFPSIRPTDLTKIPSQDNSSHLLASAYSYTFRE KSSGVQESSHFLQGAKRNNLSLAILTLEMIRNQEKVGSLGTSATNSVMYK KLENTVLLKPGLPEASGKVELLPKVHIHQEDLFPTETSNGSPGHLDLMEE ILLQKTQGAIKWNKTNRPGKVPFLKGATESSEKTPSKLLGPLAWDNQYAT QIPKEEWKSQEKSPKNTAFKTKDTILSLNPCENNHAIAAINEGQDRPQRE ATWAKQGGTGRLCSQNPPVLKRHQREITLTTLQSEQEEIDYDDTISIETK REDFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHVLRNRAQ SGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMV TFKNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM APTKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICHTNTLNPAHGRQVTV QEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAING YVMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKM AVYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSKQCQT PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKV DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTL MVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNS CSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQV NNPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTL FLQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLEV LGCEAQQLY Accordingly the sequences for sp-A1-A2-B-aq-A3-C1-C2:

(sp, Signal Peptide, SEQ ID NO: 21) MQIELSTCFFLCLLQFSFS (A1 SEQ ID NO: 22) ATRRYYLGAVELSWDYMQSDLLSELHVDTRFPPRVPRSFPFNTSVMYKKT VFVEFTDHLFNIAKPRPPWMGLLGPTIRAEVYDTVVITLKNMASHPVSLH AVGVSYWKASEGAEYDDQTSQREKEDDKVFPGESHTYVWQVLKENGPMAS DPPCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKERTQTLHQFVLLF AVFDEGKSWHSETKDSLTQAMDSASARAWPKMHTVNGYVNRSLPGLIGCH RKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEEEDYDDD LYDSEMDVVRFDDDNSPPFIQIRS (A2 SEQ ID NO: 23) VAKKHPKTWVHYIAAEEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPY NIYPHGITDVSPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTK SDPRCLTRYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNV ILFSVFDENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPF SGETVFMSMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTY EDIPTYLLSENNVIEPR (B SEQ ID NO: 24) SFSQNSRHPSTRQKQFKATTIPENDIEKIDPQFGERTQMLKVQSVSSSDL LMLLGQSPTPHGLSLSDLQEATYEAIPDDHSPGAIDSNEGPSEVAHLRPE LHHSGDMVFTPEPGLQLRLNENLGTTIAVELKKLDLKVSSSSNNLMTSPT IPSDNLAAGTEKTGSLGPPNMPVHFSSQLGTTVFGKKSSPLIGSGVPLNL SERNNDSKLLEAALMNSQESSLGKNVSSMESDRLFKEKRVHGPALLTKDN ALFKVNISLVKTNKTSNNSTTNRKTHIDGPTLLIENSTSVWQDIILESDT EFQEVTSLIHDEMLMDKNTTALRLNHVSNKTTSSKNMEMVHQKKEGPVPP DAENPDMSFFKMLFLPESANWIKRTHGKNSLNSGQGPSPKQLISLGSEKS VKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNSRSIFLTNLANVQENDT HNQEKKFQEEIERKETLIQENVVLPQVYTVTGTKNFLKNLFLLSTRQNVE GLDEGTYAPVLQDTRSLNDSTNRAEIHMAHFSKRREEENLEGLGNQTKQM VEKYPSTTRMSPNPSQQNVITQRGKRALKQFRLPLEETELEKGLIVDDTS TQWSKNMKYLTQSTLTQIDYNEKEKKAITQSPLSDCSMRNHSITQTNSSA LPIAKVSAFPSIRPTDLTKIPSQDNSSHLLASAYSYTFREKSSGVQESSH FLQGAKRNNLSLAILTLEMIRNQEKVGSLGTSATNSVMYKKLENTVLLKP GLPEASGKVELLPKVHIHQEDLFPTETSNGSPGHLDLMEEILLQKTQGAI KWNKTNRPGKVPFLKGATESSEKTPSKLLGPLAWDNQYATQIPKEEWKSQ EKSPKNTAFKTKDTILSLNPCENNHAIAAINEGQDRPQREATWAKQGGTG RLCSQNPPVLKRHQR (ap SEQ ID NO: 25) EITLTTLQSEQEEIDYDDTISIETKREDFDIYGEDENQGPR (A3 SEQ ID NO: 26) SFQKRTRHYFIAAVERLWDYGMSRSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSL ISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDMHSGLIGPLLICHTNTLNPAHGRQVTVQEFALFFTIFDETKSW YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYVMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEML PSKAGIWRVECLIGEHLHAGMSTLFLVYSK (C1 SEQ ID NO: 27) QCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNS TGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGC DLN (C2 SEQ ID NO: 28) SCSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQ VNNPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWT LFLQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLE VLGCEAQQLY

The sequence for Node number 50 is: SEQ ID NO: 29. The sequence for Node number 52 is: SEQ ID NO: 30. The sequence for Node number 57 is: SEQ ID NO: 31. The sequence for Node number 59 is: SEQ ID NO: 32. The sequence for Node number 65 is: SEQ ID NO: 33. The sequence for Node number 76 is: SEQ ID NO: 34. The sequence for Node number 78 is: SEQ ID NO: 35. The sequence for Node number 87 is: SEQ ID NO: 36. The sequence for Node number 90 is: SEQ ID NO: 37. The sequence for Node number 95 is: SEQ ID NO: 38.

SEQ ID NO: 29 MRIELPCCFFLCLLPFSFGATRRYYLGAVELSWDYVHSDLLSVLHVPTGF PGRVGRKFPFSTSVRYKKTVFVEFTDHLFTIAKPRPPWMGLLGPTIRAEV YDTVVVTLKNLASHPFSLHAVGVTYWKASEGAGYDDETSQREKEDDKVDP GKTHTYVWEVLKDNGPTASDPPCLTYSYLSYVDLVKDLNSGLIGALLVCR PGALAKDGTQTLHEFVLLFAVFDEGKSWYPEPNESLTQALDTGTPRPWPK LHTVNGYVNGSLPGLIGCHKRPVYWHVIGLGTTPEVHSIFLEGHTFLVRN HRHATLEISPITFLTAQTLLTDLGRFLLFCHIPAHQHDGMEAYVKVDVCP EEPKLRMRADAPEEDYYDDLYDLDMDVIRFDDDDSPPFIGVRAFAKKHPK TWIHYIAAEEVDWDYAPIVPTYLDRSYKSQYLEAGPQRIGRKYKKVRFVA YTDGTFKTRKVIQYDTGILGPLLYGEVGDTLLIVFKNLASRPYNIYPHGL TSVSPLHPGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTGSDPRCLT RYYYSFINPVRDLASGLIGPLLICYKESVDQRGNQMMSDKRRFILFSVFD ENRSWYLTENIQRFCPDAAGVQPQDPEFYASNVMHSINGYVFDNLHLKLC LHEVAYWYVLSVGAQTDFLSVFFSGYTFKHKMVFEDTLTLFPFSGETVFM SMEKPGLWVLGCLNPDFRDRGMWALLKVSSCDRDPGLYYGDDYDLIPTYL LNENNVLEPRGFPKNKRWPRLCLRKFKAVTSPEKDIEKLDPQFGERTQCL RAQPLKFSDLLMLLGQNPDPHGLSLSDLTEATYEPIPYDSFPGPVGNSTG PSEVVHLRPELHHAGDAGFYPEGGLLLFGKERLGPTVAVELKKLDLKLHK SVDNVMFSPTAPLDNLEKGLKGTGTLGPLKEPVRFSGHLGPTVFGKKETG LIGSGVPLQLSDRDHDSRLLERVLLNDEESPLGLNVTSLKPDRPFKDKRV HGPASLTKDNALFKGNITLVKLDKTPDNTTTNRKTYVDGPALLKENGTPV WLDILENDTRFQEVASLGFYETFQDGKLTAPGLDHVLNKTTSSKNGEVFF EKKVEPVPLDAERPPAPFFKDLFLPRTANWLLDTHGKADLRAGQGPSPKQ LISLGSDKSGDGEHFLSAKSKVRLGPDKFTKGTGLLEMIFPGRKDIFLTS LVTVQENDTLILGKKFQEAIERKETLIQENVDLPKLYVVAGTRNFLKDLF LLHTRTNLTLDGPDEGDYAPLLQDTRYLNGKVPPAGVHLVHGSKLLEAAD LEGLLHKTKRMVLDYPSTTRTAPKPCDQNCITHRGKRALKQFGLPLEETE LERGLVDKDRALKGRKNMGYLTGGTLTKIDYNDRAKRLVARPPLVDCTAR DHGVTGTDGPAGPIRGVSAFPPIRPTDLTKLPSHDNSSHLPAAACGYTFR EKSSGVRGGSHFLQGAKRTSLTSAYLTLKTIRGEEKVSSLGTSADTPPTY KKLENTVLLKPGLPEVVGKVEGLPKPHVWEADLFPTPTGNGTPGHLDLKE EWLLQKLQGAIKLSKVKRPGDFPFLKGATEILEKRPSKLLGPLPWDGQYL TQILRDEWKLKHKSPKNTVFKTKDAILPLSGCENVHGVDGINEGRDRLQK AATWAKQGGTERLCSREPPVLKNHKREITLTTLQPEKEKIDYDDYLKPET DRDDFDIYGEDENQGPRTFTGRTRHYFIAAVERLWDYGMSRPPHFLRNRA RGGRVPFFKKVVFRGFLDGSFTQPLYRGELDEHLGLLGPYIRAEVDDVIM VTFKNLASRPYSFYSSLLPYEGGLGQGSEPRKEVVKPGELRTYFWKVLPH MAPTVDEFDCKAWAYFSDVDLEKDLHSGLVGPLLICRPGTLSPAFGRQLT VQEFALLFTIFDETKSWYFTENMERNCPPPCQIQPDDPDFRRSYRFHAIN GYVMDTLPGLVMAQDQRVRWYLLSVGGTEDIHSVYFHGHVFTVRKKHEYR MGVYNLYPGVFGTVEMLPSKPGIWRVECLVGEHLQAGMSTLFLVYDPKCQ TPLGLASGHIRDAQITASGQYGQWAPRLARLHYSGSVNAWSTKDPFSWIK VDLLRPMILHGIKTQGARQKFSSLYISQFIIFYGLDGKRWKRYRGNATGT LMVFFGNVDATGVKHNRFNPPIIARYIRLHPTHYSIRTTLRMELIGCDLN SCSMPLGMENKAISDAQITASSYFTNMFATWAPSQARLHLQGRTNAWRPK VNSPKEWLQVDFEKTMRVTGIVTQGAKSLLTSMYVKEFLVSSSQDGHRWT PFLQDGKVKVFKGNQDHFTPVVNSLDPPLLTRYLRIHPRRWVHHIALRLE FLGCDAQQLY SEQ ID NO: 30 MQIELSTCFFLCLLPFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVIP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHEFVLLFAVFDEGKSWHSETNESLTQAMDSASARAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSDMDVVRFDDDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPNDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTYL LNENNVIEPRSFSQNSRHPSTRQKQFKATTTPENDIEKIDPQFGERTQLL KVQSVSSSDLLMLLGQSPTPHGLSLSDLQEATYEAIPDDHSPGATESNEG PSEVAHLRPELHHSGDMVFTPEPGLQLRLNENLGTTITVELKKLDLKVSS SSNNLMTSPTIPSDNLAAGTEKTGSLGPPNMPVHFSSQLGTIVFGKKSSP LIGSGVPLNLSERDNDSKLLEAALMNSQESSLGKNVSSMESDRLFKEKRV HGPASLTKDNALFKVNISLVKTNKTPNNSTTNRKTHIDGPTLLIENSTSV WQDILENDTEFQEVTSLIHDEMFMDKNTTALGLNHVSNKTTSSKNMEMVH QKKEGPVPLDAENPDMSFFKMLFLPDSANWIKRTHGKNSLSSGQGPSPKQ LISLGSEKSVKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNSKSIFLTN LANVQENDTHNQEKKFQEEIERKETLIQENVVLPQVYTVTGTKNFLKNLF LLSTRQNVTVEGLDEGTYAPVLQDTRSLNDSANRAGIHMAHFSKRREEAE TNLEGLGNQTKQMVEKYPSTTRMSPNPSQQNVITQRGKRALKQFRLPLEE IELERGLIVNDTSTQWSKNMKYLTQGTLTQIDYNEKEKRAITQSPLSDCS MRNHSITQTNGSALPIAKVSAFPSIRPTDLTKIPSQDNSSHLLASACSYT FREKSSGVQESSHFLQGAKRNNLSLAILTLEMIRGQEKVSSLGTSATNPL MYKKLENTVLLKPGLPEASGKVELLPKVHVHQEDLFPTETSNGSPGHLDL MEEILLQKTQGAIKLNKVNRPGKVPFLKGATESSEKTPSKLLGPLAWDNQ YATQIPREEWKSQEKSPKNTAFKTKDTILPLNPCENNHAIAAINEGQDRP QREATWAKQGGTGRLCSQNPPVLKRHQREITLTTLQPEQEKIDYDDTISI ETKREDFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHALRN RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDN IMVTFKNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ HHMAPTKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICRTNTLNPAHGRQ VTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHA INGYVMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEE YKMAVYNLYPGVFETVEMLPSKAGIWRVECLIGEHLQAGMSTLFLVYSKQ CQTPLGMASGRIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSW IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQSYRGNST GTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCD LNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWR PQVNNPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHH WTLFLQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR LEVLGCEAQQLY SEQ ID NO: 31 MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDTRFP PRVPRSFPFNTSVVYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVY DTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPG GSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRE GSLAKEKTQTLHKFVLLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNH RQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPE EPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPSVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTR YYSSFINMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCL HEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMS MENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISTYLL SKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPK VQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSL SEMTHFRPQLHHSGDMVFTPEPGLQLRLNEKLGTTAATELKKLDFKVSST SNNLISTIPSDNLAAGTDNTSSLGPPNMPVHYDSQLDTTLFGKKSSPLIE SGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKEKRAHGP ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQN ILESDTEFQKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKK EGPIPPDAENPDMSFFKMLFLPESANWIQRTHGKNSLNSGQGPSPKQLVS LGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDN LHENNTHNQEKKIQEEIERKETLIQENVVLPQIHTVTGTKNFMKNLFLLS TRQNVEGSYEGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLG NQTKQIVEKYPRTTRISPNPSQQNFVTQRGKRALKQFRLPLEETELEKRI IVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIT QANRSPLPIAKVSSFPSIRPIDLTRVLFQDNSSHLPAPSYRKKDSGVQES SHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLL KPGLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQETEG AIKWNEANRPGKIPFLRVATESSAKTPSKLLGPLAWDNHYGTQIPKEEWK SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGG TERLCSQNPPVLKRHQREITLTTLQSDQEEIDYDDTISVEMKKEDFDIYD EDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFK KVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASR PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFD CKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPG VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGH IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQ VDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQEL Y SEQ ID NO: 32 MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFP PRVPKSFPFNTSVVYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVY DTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPG GSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRE GSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNH RQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPE EPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTR YYSSFINMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDE NRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCL HEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMS MENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLL SKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPK VQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSL SEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTE SGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQN ILESDTEFQKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKK EGPIPPDAENPDMSFFKMLFLPESASWIQRTHGKNSLNSGQGPSPKQLVS LGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDN LHENNTHNQEKKIQEEIERKETLIQENVVLPQIHTVTGTKNFMKNLFLLS TRQNVEGSYEGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLG NQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRI IVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIP QANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQES SHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLP KPGLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQETEG AIKWNEANRPGKIPFLRVATESSAKTPSKLLGPLAWDNHYGTQIPKEEWK SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGR TERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYD EDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFK KVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASR PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFD CKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPG LVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGH IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQ VDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTLFFQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDL Y SEQ ID NO: 33 MQIELSTCFFLCLLQFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRF PPRMPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIWAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQMLHQFVLLFAVFDEGKSWHSETKGSLTQAMDSASARAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLLDSEMDVFRFDDDNSPPFIQIRSVAKKHPK TWIHYIAAEEEDWDYAPSVLTSDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTRETIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTYL LSENNVIEPRSFSQNSRHPSTRQKQFKATTIPENDIEKIDPQFGERTQML KVQSVSSSDLLMLLGQSPTPHGLSLSDLQEATYEAIPDDHSPGAIDSNEG PSEVAHLRPELHHSGDIVFTPEPGLQLRLNENLGTTIAVELKKLDLKVSS SPNNLMTSPTIPSDNLAAGTEKTGSLGPPNMPVHFSSQLSTTVFGKKSSP LIGSGVPLNLSERNNDSKLLEAALMNSQESSLGKNVSSMESDRLFKEKRV HGPALLTKDNALFKVNISLIKTNKTSNNSTTNRKTHIDGPTLLIENSTSV WQDTILESDTEFQEVTSLIHDEMLMDKNTTALRLNHVSNKTTSSKNMEMV HQKKEGPVPPDAENPDMSFFKMLFLPESANWIKRTHGKNSLNSGQGPSPK QLISLGSEKSVKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNNRSIFLT NLANVQENDTHNQEKKFQEEIERKEALIQENVVLPQVYTVTGTKNFLKNL FLLSTRQNIGLDEGTYAPVLQDTRSLNDSTNRAEIHMAHFSKRREEEETN LEGLGNQTKQMVEKYPSTTRMSPNPSQQNVITQRGKRALKQFRLPLEETE LEKGLIVDDTSTQWSKNMKYLTQSTLTQIDYNEKEKKAITQSPLSDSPMR NHSITQMNSSALPIAKISAFPSIRPTDLTKIPSQDNSSHFLASAYNYTFR EKSSGVQESSHFLQGAKRNNLSLAILPLEMIRNQEKVGSLGTSATNSVMY KKLENTVLLKPGLPEASGKVELLPKVPIHQEDLFPTETSHGSPGHLDLME EILLQKTQGAIKWNKTNRPGKVPFLKGATESSEKTPSKLLGPLAWDNQYA TQIPKEEWKSQEKSPKNTAFKTKDTILSLNPCENNHAIAAINEGQDRPQR EATWAKQGGTGRLCSQNPPVLKRHQREITLTTLQSEQEEIDYDDTISIET KREDFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHVLRNRD QSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIM VTFKNQASRPYSFYSSLISYKEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICHTNTLNPAHGRQVT VQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAIN GYVMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MAVYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSKQCQ IPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIK VDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLN SCSMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQ VNNPKEWLQVDFQKTMKVTGIITQGVKSLLTSMFVKEFLISSSQDGHHWT LFLQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLE VLGCEAQQLY SEQ ID NO: 34 MQIELSTCFFLCLLPFSFSATRRYYLGAVELSWDYMQSELLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVIP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHEFVLLFAVFDEGKSWHSETNESLTQAMDPASARAQPE MHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSDMDVVRFDDDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPNDRSYKSLYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFVNLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNADGVQPQDPEFQVSNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTSL LNENNVIEPRSFSQNSRHPSTRQKQFKATTTPENDIEKIDPQSGERTQLL KVQSVSSSDLLMLLGQNPTPHGLSLSDLQEATYEADDHLPGAIERNKGPS EVAHLRPELHHSGDRVFTPEPELQLRLNENLGTTITVELKKLDLKISSSS NNLMTSPTIPSDKLAAGTEKTGSLGPPNMPVHFSSQLGTIVFGKNSSHLI GSGVPLGLSEGDNDSKLLEAALMNSQESSLGENVLSMESDRLFKEERVHG PASLTKDNALFKVNISLVKTNKAPINSTTNRKTHIDGPTLLIENSTSVWQ DIILESNTEFQEVTSLIHDETFMDKNTTALGLNHVSNKTTSSKNMEMVHQ KKEGPVPLGAENPDTSFFKMLFLPDSANWIKRTHGKNSLSSGQRPSPKQL TSLGSEKSVKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNSKSIFLTNL ANVQENDTHNQEKKSQEEIERKEKLIQENVVLPQVYTVTGTKNFLKNLFL LSTKQNVEGLDEGTYTPILQDTRSLNDSANRAGIHMAHFSKIREEANLEG LGNQTKQMVEKYPSTTRMSPNPSQQNVITQRGKRALKQFRLPLEEIKLER GVILNDTSTQWSKNMKYLTQGTLTQIEYNEKEKRAITQSLLSDCSMRSHG IIQTNGSALPIAKVSAFPSIRPTDLTKIPSQDNSSHLLASACSYTFRERS SGVQESSHFLQGAKRNNLSLAFLTLEMIRGQEKISSLGKSATNPLMYKKL ENTVLLKPGLSEASGKVELLPKVHVHQEDSFPTKTSNGSPGHLDLMEEIF LQKTQGPVKLNKVNRPGKVPFLKWATESSEKTPSKLLGPLAWDNQYATQI PREEWKSQEKSQKNTAFKTKDTILPLDPCENNHSIAAINEGQDKPQREAT WAKQGGTGRLCSQNPPVLKRHQREITLTTLQPEEDKIDYDDTFSIETKRE DFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHALRNRAQSG SVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF KNQASRPYSFYSSLISYEEDQRQGAEPRKKFVKPNETKIYFWKVQHHMAP TKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICRTNTLNPAHGRQVTVQE FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYV MDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAV YNLYPGVFETVEMLPSKAGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPL GMASGRIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDL LAPMIIHSIMTQGARQKFSSLYISQFIIMYSLDGKKWQSYRGNSTGTLMV FFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCS MPLGMENKAISDAQITASSYLNNMFATWSPSQARLHLQGRTNAWRPQVNN PKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGENWTLFL QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWAHQIALRLEVLG CEAQQLY SEQ ID NO: 35 MQIELSTCFFLCLLPFSFSAIRRYYLGAVELSWDYMQSELLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDQLFNIAKPRPPWMGLLGPTIQAEV YDTVVITLKNMASHPVSLHAVGVSYWKSSEGAEYEDQTSQREKEDDKVIP GKSHTYVWQVLKENGPTASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLTKERTQTLHEFVLLFAVFDEGKSWHSGKNESLTQAMDPASARAQPA MHTINGYINRSLPGLIGCHKKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSDMDVVRFDGDNAPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTSNDRSYKSLYLNNGPQRIGRKYKKVRFIA YTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRFPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFVNLEKDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENQSWYLTENIQRFLPNADGVQPQDPEFQVSNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVYSCDRNTGDYYEDTYEDIPTFL LNENNVIEPRSFSQNSRHPSTRQKQFKATTTPENDIEKIDPQSGERTQLL KEQSVSSSDLLMLLGQNPTPHGLSLSDLQEARNEADDHLPGAIERNKGPS EVAHLRPELHHSGERVFTPEPELPLRLNENLGTTITVELKKLDFKISSSS NNLMTSPTIPSDKLSAGTEKTGSLGPPNMPVNFSSQLGTIVFGKNSSHFI GSGVPLGLSEEDNDSKLLEAALMNSQESSLGENVLSMESDRLFKEERVHG PASLTKDDALFKVNISLVKTNKAPVNSTTNRKTHIDDPTLLIENSTSVWQ DIILESNTEFQEVTSLIHDETFMDKNTTALGLNHVSNKTTSSKNMEMVHQ KKEGPVPLDAEYPDTSFFKTLFLPDSTNWIKRTHGKNSLSSGQRPSPKQL TSSGSEKSVKDQNFLSEKNKVVVGEDEFSKDTGLKEMIFPNSKSIFLTNL ANVQENDTHNQEKKSQEEIERKEKLIQENVVLPQVYTVTGTKNFLKNLFL LSTKQNVEGLDEGTYTPVLQDTRSLNDSAKRAGIHMAHFSKIREEANLEG LGNQTKQMVEKYPSTTRMSPNPSQQNVIPQRGKRDLKQFRLPLEEIKLER GVILNDTSTQWSKNMKYLTQGTFTQIEYNKKEKRAITQSFLSDCSMRSHG IIQTNGSALPIAKVSAFPSIRPTDLTKIPSQDNSSHLPASACSYTFGERS SGVQESSHFLQGAKRNNLSLAFLTLEMIRGQGKISTLGKSATNPLMYKKL ENTVLLKPGLSEASGKVEFLPKVHVHQEDFFPTKTSNGSPAHLDLREEIF LQKTQGLVKLNKVNRPGKVPFLKWATESSEKTPSKLLGPLAWDNQYATLI PREEWKSLEKSQKNTAFKTKDTILPLDPCENNHSIAAINEGQDKPQREAT WVKQGGTGRLCSQNPPVLKRHQREITLTTFQPEEDKIDYDDTFSIETKRE DFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHALRNRAQNG DVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF KNQASRPYSFYSSLISYEEDQRQGAEPRKKFVKPNETKIYFWKVQHHMAP TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRTNTLNAAHGRQVTVQE FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYV MDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAV YNLYPGVFETVEMLPSKVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPL GMASGRIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDL LAPMIIHSIMTQGARQKFSSLYISQFIIMYSLDGKKWQSYRGNSTGTLMV FFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCS MPLGMENKAISDAQITASSHLSNMFATWSPSQARLHLQGRTNAWRPQVNN PKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHNWTLFL QNGKVKVFQGNQDSFTPVVNALDPPLFTRYLRIHPQSWAHHIALRLEVLG CEAQQLY SEQ ID NO: 36 MQIELSTCFFLCLLPFSFSATRRYYLGAVELSWDYMQSELLSELHVDTRF SPRVPRSLPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQKEKEDDKVIP GESHTYVWQVLKENGPMASDPPCLTYSYFSHVDLVKDLNSGLIGALLVCK EGSLAKERTQTLHEFVLLFAVFDEGKSWHSETNESLTQSAQAQHEMHTVN GYVNRSLPGLTGCHKKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS LEISPITFLTAQTFLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQL RMKNNEEEEDYDDDLYDSDMDVVRFDGDNSSPFIQIRSVAKKHPKTWVHY IAAEEEDWDYAPSAPTPNDRSYKNLYLNNGPQRIGRKYKKVRFMAYTDET FKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSP LHSGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS FINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSW YLTENMQRFLPNADAVQPHDPEFQVSNIMHSINGYVFDNLQLSVCLHEVA YWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWVLGCHNSDFRNRGMTALLKVSSCNRNTGDYYEDTYEDIPTSLLNENN VIEPRSFSQNSRHPSTRQKQFKATTTPENDIEKIDPQSGERTQLLKVQSV SSSDLLMLLGQNPTPHGLFLSDLQEATHEADDHLLGAIERNKGPSEVASL RPELHHSGDRVFTPEPELQLRLNENLGTTVTVELKKLDLKISSSSDNLMT SPTIPSDKLAAGTEKTGSLGPPNMSVHFNSHLGTIVFGKNSSHLIESGVP LGLSEGDNDSKLLEAALMNSQESSLGENVLSMESNRLFKEERVHGPASLI KDNALFKVNISLVKTNKAPINSTTNRKTHVDVPTLLIENSTSVWQDIILE SNTEFQEVTSLIHNETFMDRNTTALGLNHVSNKTTSSKNVEMVHQKKEGP VPLGAENPDLSFFKILFLPDSANWIKRTHGKNSLSSGQRPSPKQLTSLGS EKSVKDQNFLSEKKVVVGEDEFTKDTGLKEMIFPNSKSIFFTNLANVQEN DTYNQEKKSQEEIERKEKLTQENVVLPQVYTVTGTKNFLKNLFLTKQNVT VEGLDEGPYTPILQDTRSLNDSAHRAGIHMAHFSKIREEANLEGLGNQTK QMVERFPSTTRMSPNPSQHNVITQRGKRALKQPRLSLEEIKFERGVILND TSTQWSKNMNYLTQGTLTQIEYNEKEKRAITQSLLSDCSMRNHGTIQMND SALPIAKVSAFPSIRHTDLTKIPSQDNSSHLPASACSYTFRERSSGVQES SHFLQGAKRNNLSLAFLTLEMIRGQGKFSSLGKSATNQPMYKKLENTVLL KPGLSEASGKVELLPKVHVHQEDSFPTKTSNDSPGHLDLMEKIFLQKTQG PVKLNKVNRPGKVPFLKWATESSEKIPSKLLGPLAWDNHYATQIPREEWK SQKKSQKNTAFKTKDTILPLGPCENNHSIAAINEGQDKPQREATWAKQGE TGRLCSQNPPVSKRHQREITLTTLQPEEDKIEYDDTFSTEMKREDFDIYG EDENQGLRSFQKRTRHYFIAAVERLWDYGMSRSPHALRNRAQSGDVPQFK KVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASR PYSFYSSLISYEEDQRQGAEPRKKFVNPNETKIYFWKVQHHMAPTKDEFD CKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQKEDPTFKENYRFHAINGYVMDTLPG LVMAQDQKIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPG VFETVEMLPSKVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPLGMASGH IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDLLAPMII HSIMTQGARQKFSSLYISQFIIMYSLDGKKWQSYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME NKAISDAQITASSYLNSMFATWSPSQARLHLQGRTNAWRPQANNPKEWLQ VDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGENWTLFLQNGKVK VFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWAHQIALRLEVLGCEAQQL Y SEQ ID NO: 37 MQIELSTCFFLCLLPFSFSATRRYYLGTVELSWDYMQSELLSELHVDTRF PPRVPRSFPFNPSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVIP GESYTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHEFVLLFAVFDEGKSWHSETNESLTQAMDPASARAQPE MHTVNGYVNRSLPGLIGCHKKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPISFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSDMDVVRFDGDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPNDRSYKSLYLNNGPQQIGRKYKKARFMA YTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFVNLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNADGVQPQDPEFQVSNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCNRNTGDYYEDTYEDIPTSL LNENNVIEPRSFSQNSRHPSTRQKQFKATTTPENDVEKIDLRSGERTQLL KVQSVSSSDLLMLLGQNPTPHALSLSDLQEVTYEADDHLPGTIERNKGPS EVAHLRPELHHSGDRVFTPEPELQLRLNENLGTTITVELKKLDLKISSSS NNLMISPTIPSDKLAAGTEKTGSLGPPNMPVHFSSQLGTIVFGKNSSHLI ESAVPLGLSEGDNDSKLIEAALMNSQESSLEENVLSMESDRLFKEERVHG PVSLTKDNALFKVNFSLVKTNKAPINSTTNRKTHIDGPTLLIENSTSVWQ DIILESNSGFQEVTSLIHDETFMDKNTTALGLNHVSNKTTSSKNMEMVHQ KKEDPAPLGAENPDISFFKMLFLPDSANWIKRTHCKNSLSSGQRPSPKQL TSLGSEKSVKDQNFLSEKNKVVVGEDEFTKDTGLKEMIFPNSKSIFLTNL ANVQENDTHNQEKNSQEEIERKEKLIQKNVVLPQVYTVTGTKNFLKNLFL LSTKQNVEGLDEGTYTPILQDTRSLNESANRARIHMAHFSKIREEANLEG LGNQTKQMVEKYPSTTRMSPNPRQQNVITHHGKRALKQFRLPQEEIKLER GVILNDTSTQWSKNMKYLTQGTLTQIEYNEKEKRAITQSLLSDCSVRSHG IIQTNGSALPIAKVSAFPSIRPTDLTKIPSQDNSSHLLASACSYTFREKS SGIQESSHFLQGAKRNNLSLAFLTLEMIRGQGKISSLGKLSEASGKVELL PKVHVHQEDSFPTKTSNGSPGHLDLMEEIFLQKTQGPVKLNKVNRPGKIP FLKWAAESSEKTPSKLLGPLATQIPREEWNSQEKSQKNKAFKTKDTISPL DPCENNHSIAAINKGQDKPQREATWAKQEETGRLCSQNPPVLKRHQRQIT LTTVQPEEDKIDYDDTFSTETKREDFDIYGEDENQDPRSFQKRTRHYFIA AVERLWDYGMSRSPHALRNRAQSGDVPQFKKVVFQEFTDGSFTQPLYRGE LNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYEDDQRQGAE PRKKFVKPNETEVYFWKVQHHMAPTKDEFDCKAWAYFSDVDLDKDVHSGL VGPLLICRANTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYVMDTLPGLVMAQDQRIRWYLLSMGSNE NIHSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSKVGIWRIECL IGEHLQAGMSTLFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLA RLHYSGSINAWSTKDPFSWIKVDLLAPMIIHSIMTQGARQKFSSLYISQF IIMYSLDGKKWQSYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMENKAIADAQITASSYLNNMFA TWSPSQARLHLQGRTNAWRPRVNNPKEWLQVDFQKTMKVTGITTQGVKSL LTSMYVKEFLISSSQDGHNWTLFLQNGKVKVFQGNQDSFTPVVNSLDPPL LTRYLRIHPQSWGHQIALRLEVLGCEAQQLY SEQ ID NO: 38 MQIELSVCFFLCLLPFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRL LPRVPRSFPFNTSVMFKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVIITLKNMASHPVSLHAVGVSYWKASEGAEYDDKTSQREKEDDKVIP GESHTYIWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHEFVLLFAVFDEGKSWHSEANESLAQGVDSASTRPWPK MYTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHIPSHQHDGMEAYVKVDSCP EEPQLRKKNNEEEEDYEDDLYDSDMDVLRFDDDNSPPFIQIRSVAKKHPK TWIHYIAAEEEDWDYAPAVLPSTDRSYKSQYLNNGTQRIGRKYKKVRFIA YTDETFQTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TNVSPLHSGRLPKGVKHLKDLQIMPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNAAGVQPQDPEFQASNMMYSINGYVFDNLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVFEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTDDYYEDTYEDIPTYL LNENSVIEPRSFSQNSRHPSPRQKQFKATTAPENDIEKMDPRFGERTQLL KAQSVSSSDLLMLLGQSPTPHGLSLSDLQEAKYEAIPDDPSPGAIENKEG PSEVAHLRPELHHSEDMVFTPEPGLQLRLNENLETTITEDLKKLDLKVSS SSNNVMTSPISSSDNLPADTEKTDSLGPLNTPVHFSSQLGTILFGKKSSP LIGSGVPLNLSERDNDSTLLEAALMSSQESSLGKKVSSMESDRLFKEKKV HGPASLTKDNALFKVNISLVKTNKTPNNSTTNRKTHIDGPTLLNENSTSG WQDILKNDTEFQEVTSLIHNEMFMDKNTTALGLNHVSNKTTSSKDMEMVH QKKEDPVSLDAENPDMSFFKMLFLPDSANWIKRTHGKNSLSSEQGPSPKQ LISLGSENSVKDQNFLSEKNKVAVGEDEFTKDTGFKEMIFPNSKSIFLTN LANVQENDTQNQEKKFQEEIERKETLIQENVGLPQVFPVTGTKNFLKTLF LLSTRQNVTVEGLDEGTYAPVLQDTRSLNDSANRLGLHMAHFSKRREEAN LEGLRNQTKQMVEKYPSPTRMSPNPSQQNAITQRSKRALKQFGPPLEEIE LERGLIVNDTSTLQSRNMKYLTQGTLTQIDYNEKEKRAITQSPLSDCSMR NHVITQTNGSALPIAKTSAFPSIRPTDLTKIPSQDNSSHLLSSACNYTFR EKSSGVQESSHFLQGAKRNNLSSAILTLDMIRGQEKVISLSATDPLMYKK LENTVLLKPGLPEASGKVELLPKIHVHQEDPFPTETSNGSPGHLDLMEEI LLQKTQGAIKLNKVNRPGKVPFLKGATESSEKTLPKLLGPLAWDNQYATQ ISREEWQSQEKSPKNTAFNTKDTISPLNPCENNHAIAAINEGQDRLQKEA TWAKQGETERLCSENPPVLKHHPREITLTALQQEQEKIDYDDALSIETKR EDFDIYGEDENQGPRSFQKKTRHYFIAAVERLWDYGMSKSPHALRNRAQS GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FKNQASRPYSFYSSLISYEEDQRQGTEPRKNLVKPNETKTYFWKVQHHMA PTKDEFDCKAWAYFSDVDLEKDLHSGLIGPLLICRTNTLNPAHGRQLTVQ EFALFFTIFDETKSWYFTENMERNCKAPCNIQMEDPTFKKNYRFHAINGY VMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA VYNLYPGVFETVEMLPSKAGIWRVECLIGEHLQAGMSTLFLVYSKECQTP LGMASGRIRDSQITASGHYGQWAPKLARLHYTGSINAWSTKDPFSWIKVD LLAPMIIHGIKTQGARQKLSSLYISQFIIMYSLDGKKWQSYRGNSTGTLM VFFGNVDSSGIKHNIFNPPIIAQYIRLHPTHHSIRSTLRMELMGCDLNSC SMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQVN NPKEWLQVDFQKTMKVTGITTQGAKSLLTSMYVKEFLISSSQDGEIHWTL FLQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIYPQSWVHQIALRLEV LGCQSQQHY SEQ ID NO: 39 MQIALFTCFFLSLFNFCSSATRRYYLGAVELSWNYMQSDLLSVLHTDTRF LPRMPTSFPFNTSIMYKKTVFVEYMDHLFNIAKPRPPWMGLLGPTIWTEV HDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCK EGSLSKERTQMLHQFVLLFAVFDEGKSWHSETKDSFTQAMDSASTRAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCP EEPQWQKKNNEEMEDYDDDLDSEMDMFTLDDDNSPFIQIRSVAKKYPKTW IHYISAEEEDWDYAPSVLTSDDGSYKSQYLSNGPHRIGRKYKKVRFIAYT DVTFKTRETIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITD VSPLHSRRLPRGIKHVKDLPIRPGEIFKYKWTVTVEDGPTKSDPRCLTRY YSSFINPERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDEN QSWYITENMQRFLPNAADTQPQDPEFQASNIMHSINGYVFDSLQLTVCLH EVAYWYILSVGAQTDFLSIFFSGYTFKHKMVYEDTLTLFPFSGETVFMSM ENPGLWVLGCHNSDFRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQLVN DNNVIEPRSFFQNPPVLKRHQRELSALQSEQEATDYDDSITIETNEDFDI YGEDIKQGPRSFQQKTRHYFIAAVERLWDYGMSTSPHVLRNRDQSGNAPQ FKKVVFQEFTDGSFSQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQA SRPYSFYSSLISYKEDQRQGEEPRRNFVKPNETKIYFWKVQEIHMAPTED EFDCKAWAYFSDVDLERDMHSGLIGPLLICHTNTLNPAHGRQVAVQEFAL FFTIFDETKSWYFTENVERNCKTPCNIQMEDPTLKENYRFHAINGYVMDT LPGLVMAQDQRIRWYLLSMGSNENIQSIHFSGHVFTVRKKEEYKMAVYNL YPGVFETVEMLPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMA SGSIRDFQITASGHYGQWAPNLARLEIHSGSINAWSTKEPFSWIKVDLLT PMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWLSYRGNSTGTLMVFF GNVDSSGIKHNSFNPPIIARYIRLHPTHSSIRSTLRMELMGCDLNSCSIP LGMENKVISDTQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQVNDPK EWLQVDLQKTMKVTGIITQGVKSLFTSMFVKEFLISSSQDGEIHWTHILH NGKVKVFQGNQDSSTPMVNSLDPPLLTRYLRIHPQIWEHQIALRLEILGC EAQQLY SEQ ID NO: 40 MQIELSTCFFLCLLQFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHQFVLLFAVFDEGKSWHSETKDSLTQAMDSASARAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSEMDVVRFDDDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDVTFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI TDVSPLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLT RYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFD ENRSWYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVC LHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFM SMENPGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTYL LSENNVIEPRSFSQNPPVLKRHQREITLTTLQSEQEEIDYDDTISIETKR EDFDIYGEDENQGPRSFQKRTRHYFIAAVERLWDYGMSRSPHVLRNRAQS GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FKNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMA PTKDEFDCKAWAYFSDVDLEKDMHSGLIGPLLICHTNTLNPAHGRQVTVQ EFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGY VMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA VYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSKQCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVD LLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSC SMPLGMESKAISDAQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQVN NPKEWLQVDFQKTMKVTGITTQGVKSLLTSMYVKEFLISSSQDGHHWTLF LQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLEVL GCEAQQLY SEQ ID NO: 42: MQIALFTCFFLSLFNFCSSATRRYYLGAVELSWNYMQSDLLSVLHTDTRF LPRMPTSFPFNTSIMYKKTVFVEYMDHLFNIAKPRPPWMGLLGPTIWTEV HDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCK EGSLSKERTQMLHQFVLLFAVFDEGKSWHSETKDSFTQAMDSASTRAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCP EEPQWQKKNNEEMEDYDDDLDSEMDMFTLDDDNSPFIQIRSVAKKHPKTW VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYT DVTFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITD VRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRY YSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDEN RSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLH EVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSM ENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFAQNSRPPSASAPKPPVLKRHQRELSALQSEQEATDYDDSI TIETNEDFDIYGEDIKQGPRSFQQKTRHYFIAAVERLWDYGMSTSPHVLR NRDQSGNAPQFKKVVFQEFTDGSFSQPLYRGELNEHLGLLGPYIRAEVED NIMVTFKNQASRPYSFYSSLISYKEDQRQGEEPRRNFVKPNETKIYFWKV QHHMAPTEDEFDCKAWAYFSDVDLERDMHSGLIGPLLICHTNTLNPAHGR QVAVQEFALFFTIFDETKSWYFTENVERNCKTPCNIQMEDPTLKENYRFH AINGYVMDTLPGLVMAQDQRIRWYLLSMGSNENIQSIHFSGHVFTVRKKE EYKMAVYNLYPGVFETVEMLPSRAGIWRVECLIGEHLQAGMSTLFLVYSK KCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNS TGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGC DLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAW RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIAL RMEVLGCEAQDLY

In certain embodiments, the disclosure contemplate recombinant, chimeric, non-naturally occurring sequences comprising one or more of the sequences of four or more continuous amino acids found in Ancestral Nodes disclosed herein, e.g., 68 or 53.

In certain embodiments, the sp domain comprises one or more sequences selected from; IALF (SEQ ID NO: 44), FFLS (SEQ ID NO: 45), FLSL (SEQ ID NO: 46), LSLF (SEQ ID NO: 47), SLFN (SEQ ID NO: 48), LFNF (SEQ ID NO: 49), FNFC (SEQ ID NO: 50), NFCS (SEQ ID NO: 51), FCSS (SEQ ID NO: 52), CSSA (SEQ ID NO: 53), SSAT (SEQ ID NO: 54).

In certain embodiments, the A1 domain comprises one or more sequences selected from; LSWN (SEQ ID NO: 55), WNYM (SEQ ID NO: 56), NYMQ (SEQ ID NO: 57), DLLS (SEQ ID NO: 58), LLSV (SEQ ID NO: 59), LSVL (SEQ ID NO: 60), SVLH (SEQ ID NO: 61), VLHT (SEQ ID NO: 62), LHTD (SEQ ID NO: 63), HTDT (SEQ ID NO: 64), TDTR (SEQ ID NO: 65), TRFL (SEQ ID NO: 66), RFLP (SEQ ID NO: 67), FLPR (SEQ ID NO: 68), LPRM (SEQ ID NO: 69), PRMP (SEQ ID NO: 70), RMPT (SEQ ID NO: 71), PTSF (SEQ ID NO: 72), TSFP (SEQ ID NO: 73), NTSI (SEQ ID NO: 74), TSIM (SEQ ID NO: 75), SIMY (SEQ ID NO: 76), IMYK (SEQ ID NO: 77), MYKK (SEQ ID NO: 78), FVEY (SEQ ID NO: 79), VEYM (SEQ ID NO: 80), EYMD (SEQ ID NO: 81), YMDH (SEQ ID NO: 82), MDHL (SEQ ID NO: 83), DHLF (SEQ ID NO: 84), PTIW (SEQ ID NO: 85), TIWT (SEQ ID NO: 86), IWTE (SEQ ID NO: 87), WTEV (SEQ ID NO: 88), TEVH (SEQ ID NO: 89), EVHD (SEQ ID NO: 90), VHDT (SEQ ID NO: 91), HDTV (SEQ ID NO: 92), FPGE (SEQ ID NO: 93), PGES (SEQ ID NO: 94), GESH (SEQ ID NO: 95), ESHT (SEQ ID NO: 96), LVCK (SEQ ID NO: 97), VCKE (SEQ ID NO: 98), CKEG (SEQ ID NO: 99), GSLS (SEQ ID NO: 100), SLSK (SEQ ID NO: 101), LSKE (SEQ ID NO: 102), SKER (SEQ ID NO: 103), RTQM (SEQ ID NO: 104), TQML (SEQ ID NO: 105), QMLH (SEQ ID NO: 106), MLHQ (SEQ ID NO: 107), LHQF (SEQ ID NO: 108), HQFV (SEQ ID NO: 109), QFVL (SEQ ID NO: 110), KDSF (SEQ ID NO: 111), DSFT (SEQ ID NO: 112), SFTQ (SEQ ID NO: 113), FTQA (SEQ ID NO: 114), TQAM (SEQ ID NO: 115), QAMD (SEQ ID NO: 116), AMDS (SEQ ID NO: 117), MDSA (SEQ ID NO: 118), DSAS (SEQ ID NO: 119), SAST (SEQ ID NO: 120), ASTR (SEQ ID NO: 121), STRA (SEQ ID NO: 122), TRAW (SEQ ID NO: 123), LLID (SEQ ID NO: 124), LIDL (SEQ ID NO: 125), IDLG (SEQ ID NO: 126), SSHK (SEQ ID NO: 127), SHKH (SEQ ID NO: 128), HKHD (SEQ ID NO: 129), KHDG (SEQ ID NO: 130), EPQW (SEQ ID NO: 131), PQWQ (SEQ ID NO: 132), QWQK (SEQ ID NO: 133), WQKK (SEQ ID NO: 134), QKKN (SEQ ID NO: 135), KKNN (SEQ ID NO: 136), NEEM (SEQ ID NO: 137), EEME (SEQ ID NO: 138), EMED (SEQ ID NO: 139), MEDY (SEQ ID NO: 140), DDLD (SEQ ID NO: 141), DLDS (SEQ ID NO: 142), LDSE (SEQ ID NO: 143), EMDM (SEQ ID NO: 144), MDMF (SEQ ID NO: 145), DMFT (SEQ ID NO: 146), MFTL (SEQ ID NO: 147), FTLD (SEQ ID NO: 148), TLDD (SEQ ID NO: 149), LDDD (SEQ ID NO: 150).

In certain embodiments, the ap domain comprises one or more sequences selected from; LSAL (SEQ ID NO: 151), SALQ (SEQ ID NO: 152), ALQS (SEQ ID NO: 153), LQSE (SEQ ID NO: 154), QSEQ (SEQ ID NO: 155), SEQE (SEQ ID NO: 156), EQEA (SEQ ID NO: 157), QEAT (SEQ ID NO: 158), EATD (SEQ ID NO: 159), ATDY (SEQ ID NO: 160), TDYD (SEQ ID NO: 161), YDDS (SEQ ID NO: 162), DDSI (SEQ ID NO: 163), DSIT (SEQ ID NO: 164), SITI (SEQ ID NO: 165), ITIE (SEQ ID NO: 166), TIET (SEQ ID NO: 167), IETN (SEQ ID NO: 168), ETNE (SEQ ID NO: 169), TNED (SEQ ID NO: 170), NEDF (SEQ ID NO: 171), DIYG (SEQ ID NO: 172), IYGE (SEQ ID NO: 173), YGED (SEQ ID NO: 174), GEDI (SEQ ID NO: 175), EDIK (SEQ ID NO: 176), DIKQ (SEQ ID NO: 177), IKQG (SEQ ID NO: 178), KQGP (SEQ ID NO: 179), QGPR (SEQ ID NO: 180).

In certain embodiments, the A3 domain comprises one or more sequences selected from; MSTS (SEQ ID NO: 181), STSP (SEQ ID NO: 182), TSPH (SEQ ID NO: 183), RNRD (SEQ ID NO: 184), NRDQ (SEQ ID NO: 185), RDQS (SEQ ID NO: 186), DQSG (SEQ ID NO: 187), QSGN (SEQ ID NO: 188), SGNA (SEQ ID NO: 189), GNAP (SEQ ID NO: 190), NAPQ (SEQ ID NO: 191), APQF (SEQ ID NO: 192), GSFS (SEQ ID NO: 193), SFSQ (SEQ ID NO: 194), FSQP (SEQ ID NO: 195), SQPL (SEQ ID NO: 196), ISYK (SEQ ID NO: 197), SYKE (SEQ ID NO: 198), YKED (SEQ ID NO: 199), KEDQ (SEQ ID NO: 200), RQGE (SEQ ID NO: 201), QGEE (SEQ ID NO: 202), GEEP (SEQ ID NO: 203), EEPR (SEQ ID NO: 204), EPRR (SEQ ID NO: 205), PRRN (SEQ ID NO: 206), RRNF (SEQ ID NO: 207), RNFV (SEQ ID NO: 208), ETKI (SEQ ID NO: 209), TKIY (SEQ ID NO: 210), KIYF (SEQ ID NO: 211), IYFW (SEQ ID NO: 212), DLER (SEQ ID NO: 213), LERD (SEQ ID NO: 214), ERDM (SEQ ID NO: 215), RDMH (SEQ ID NO: 216), DMHS (SEQ ID NO: 217), MHSG (SEQ ID NO: 218), LICH (SEQ ID NO: 219), ICHT (SEQ ID NO: 220), CHTN (SEQ ID NO: 221), HTNT (SEQ ID NO: 222), TNTL (SEQ ID NO: 223), TLNP (SEQ ID NO: 224), LNPA (SEQ ID NO: 225), NPAH (SEQ ID NO: 226), PAHG (SEQ ID NO: 227), RQVA (SEQ ID NO: 228), QVAV (SEQ ID NO: 229), VAVQ (SEQ ID NO: 230), AVQE (SEQ ID NO: 231), RNCK (SEQ ID NO: 232), NCKT (SEQ ID NO: 233), CKTP (SEQ ID NO: 234), KTPC (SEQ ID NO: 235), TPCN (SEQ ID NO: 236), ENIQ (SEQ ID NO: 237), NIQS (SEQ ID NO: 238), IQSI (SEQ ID NO: 239), QSIH (SEQ ID NO: 240), LPSR (SEQ ID NO: 241), PSRA (SEQ ID NO: 242), SRAG (SEQ ID NO: 243), RAGI (SEQ ID NO: 244).

In certain embodiments, the recombinant or chimeric protein comprises one or more of the sequences found to be unique in Ancestral Nodes 53 selected from in the sp domain, CLLQ (SEQ ID NO: 247), LLQF (SEQ ID NO: 248), LQFS (SEQ ID NO: 249), QFSF (SEQ ID NO: 250). In the A1 domain LLSE (SEQ ID NO: 251), LSEL (SEQ ID NO: 252), ELHV (SEQ ID NO: 253), RVPR (SEQ ID NO: 254), VPRS (SEQ ID NO: 255), PTIR (SEQ ID NO: 256), TIRA (SEQ ID NO: 257), IRAE (SEQ ID NO: 258), NEEE (SEQ ID NO: 259), EEED (SEQ ID NO: 260), CDRN (SEQ ID NO: 261), EDTY (SEQ ID NO: 262), PTYL (SEQ ID NO: 263), LSEN (SEQ ID NO: 264). In the activation peptide domain, ITLT (SEQ ID NO: 265), SIET (SEQ ID NO: 266), IETK (SEQ ID NO: 267), ETKR (SEQ ID NO: 268), TKRE (SEQ ID NO: 269), KRED (SEQ ID NO: 270), REDF (SEQ ID NO: 271), QKRT (SEQ ID NO: 272). In the A3 domain, VEQL (SEQ ID NO: 273), EGLW (SEQ ID NO: 274), GMSR (SEQ ID NO: 275), MSRS (SEQ ID NO: 276), SRSP (SEQ ID NO: 277).

Ancestral Protein Reconstruction

Recombinant porcine FVIII was approved by the FDA for the treatment of hemophilia A patients who have developed an autoimmune response to endogenous FVIII. Porcine FVIII displays reduce cross-reactivity toward antibody inhibitors developed against human FVIII. It is desirable to find FVIII sequences that have improved activity because it is possible that the frequency of infusion may be lessened while still achieving full prophylaxis. Murine FVIII (mFVIII) has displayed the longest observed half-life following activation.

A phylogenetic tree was constructed based on known orthologous FVIII sequences. Ancestral FVIII sequences were reconstructed and expressed in multiple cell lines. The ancestral FVIII sequences produced functional proteins that complexed with human coagulation factors. A sequence for the common ancestor of primates and rodents demonstrates higher biosynthesis/secretion than more evolved primates. Several ancestral FVIII sequences are expressed at greater levels than human FVIII in transient and recombinant expression systems.

Recombinant Proteins with the B domain deleted were produced.

The B-domain deleted sequence for Node number 68 having the E434V mutation is: SEQ ID NO: 39

MQIALFTCFFLSLFNFCSSATRRYYLGAVELSWNYMQSDLLSVLHTDTRF LPRMPTSFPFNTSIMYKKTVFVEYMDHLFNIAKPRPPWMGLLGPTIWTEV HDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCK EGSLSKERTQMLHQFVLLFAVFDEGKSWHSETKDSFTQAMDSASTRAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCP EEPQWQKKNNEEMEDYDDDLDSEMDMFTLDDDNSPFIQIRSVAKKYPKTW IHYISAEEEDWDYAPSVLTSDDGSYKSQYLSNGPHRIGRKYKKVRFIAYT DV [E434V mutation] TFKTRETIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVS PLHSRRLPRGIKHVKDLPIRPGEIFKYKWTVTVEDGPTKSDPRCLTRYYS SFINPERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENQS WYITENMQRFLPNAADTQPQDPEFQASNIMHSINGYVFDSLQLTVCLHEV AYWYILSVGAQTDFLSIFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMEN PGLWVLGCHNSDFRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQLVNDN NVIEPRSFFQN [B-domain deletion] PPVLKRHQRELSALQSEQEATDYDDSMETNEDFDIYGEDIKQGPRSFQQK TRHYFIAAVERLWDYGMSTSPHVLRNRDQSGNAPQFKKVVFQEFTDGSFS QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKE DQRQGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLE RDMHSGLIGPLLICHTNTLNPAHGRQVAVQEFALFFTIFDETKSWYFTEN VERNCKTPCNIQMEDPTLKENYRFHAINGYVMDTLPGLVMAQDQRIRWYL LSMGSNENIQSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSRAG IWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMASGSIRDFQITASGHYG QWAPNLARLHHSGSINAWSTKEPFSWIKVDLLTPMIIHGIKTQGARQKFS SLYISQFIIMYSLDGKKWLSYRGNSTGTLMVFFGNVDSSGIKHNSFNPPI IARYIRLHPTHSSIRSTLRMELMGCDLNSCSIPLGMENKVISDTQITASS YFTNMFATWSPSQARLHLQGRTNAWRPQVNDPKEWLQVDLQKTMKVTGII TQGVKSLFTSMFVKEFLISSSQDGFIHWTHILHNGKVKVFQGNQDSSTPM VNSLDPPLLTRYLRIHPQIWEHQIALRLEILGCEAQQLY The B-domain deleted sequence for Node number 53 having E434V mutation is: SEQ ID NO: 40

MQIELSTCFFLCLLQFSFSATRRYYLGAVELSWDYMQSDLLSELHVDTRF PPRVPRSFPFNTSVMYKKTVFVEFTDHLFNIAKPRPPWMGLLGPTIRAEV YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCR EGSLAKERTQTLHQFVLLFAVFDEGKSWHSETKDSLTQAMDSASARAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP EEPQLRMKNNEEEEDYDDDLYDSEMDVVRFDDDNSPPFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPSVLTPDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDV [E434V mutation] TFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVS PLHSGRLPKGVKHLKDLPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYS SFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRS WYLTENMQRFLPNAAGVQPQDPEFQASNIMHSINGYVFDSLQLSVCLHEV AYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMEN PGLWVLGCHNSDFRNRGMTALLKVSSCDRNTGDYYEDTYEDIPTYLLSEN NVIEPRSFSQN [B-domain deletion] PPVLKRHQREITLTTLQSEQEEIDYDDTISIETKREDFDIYGEDENQGPR SFQKRTRHYFIAAVERLWDYGMSRSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSL ISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDMHSGLIGPLLICHTNTLNPAHGRQVTVQEFALFFTIFDETKSW YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYVMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEML PSKAGIWRVECLIGEHLHAGMSTLFLVYSKQCQTPLGMASGHIRDFQITA SGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNI FNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ ITASSYFTNMFATWSPSQARLHLQGRTNAWRPQVNNPKEWLQVDFQKTMK VTGITTQGVKSLLTSMYVKEFLISSSQDGFIHWTLFLQNGKVKVFQGNQD SFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRLEVLGCEAQQLY Biosynthetic Rates of Ancestral FVIII Molecules

Constructs within the ancestral mammalian Orders—Primates, Rodentia and Perissodactyla were reconstructed through gene transfer and expression in human embryonic kidney (HEK) 293 and baby hamster kidney (BHK) mammalian cell lines. Each construct displayed detectable FVIII activity as determined by one-stage coagulation assay. This assay utilizes human FVIII-deficient plasma in combination with a test substance to generate a macroscopic clot capable of physically trapping a metal ball being pulled by magnetic forces. This assay also confirmed the compatibility of each An-FVIII with human coagulation factor IXa, for which FVIII serves as a cofactor, as well as factor X, the substrate of factor IXa. Following transient transfection of HEK293T-17 cells with plasmid DNA encoding the An-FVIII variants, 11-, 9-, and 4-fold enhanced FVIII activity was observed from the cells expressing the inferred primate ancestors An-54, -53, and -52, respectively, relative to existing human FVIII. However, the closest inferred An-57 shares 99% identity to hFVIII and did not display a significant difference in activity. Within the non-primate groups, An-68 demonstrated the greatest activity at 14-fold relative to hFVIII while An-65, -78, and -76 did not display different secreted activity concentrations. Of note, porcine FVIII (pFVIII) and the human porcine hybrid ET3 demonstrated 8 and 7-fold increased activity, respectively.

In Vivo ED₅₀ Determination of Recombinant an-FVIII

To study the in vivo efficacy of ED₅₀ of two An-FVIII sequences, An-53 and An-68, secreted protein was purified to homogeneity using two-step ion exchange FPLC. Purity of the protein was confirmed using SDS-PAGE, and specific activities of An-53 and An-68 were measured as 18,500 and 8770 U/mg, respectively. Hemophilia A E16 mice of 8-12 weeks were administered a single dose of FVIII and challenged via tail transfection 15 minutes following injection. For the Dixon up-and-down method, doses were determined a priori based upon previous investigation of human FVIII ED₅₀. A bleeding event was defined as blood loss exceeding the mean plus standard deviation of wildtype C57BL/6 mice following the same challenge. Completion of this study revealed that An-53 and An-68 yielded ED₅₀ values of 88.6 and 47.2 U/kg, respectively. The resulting efficacies mirror the differences in specific activities.

TABLE 1 ED50 determination of An-53 and An-68 by Dixon up-and-down method via murine tail transection bleeding challenge. Dixon Up-and-Down Method for Small Samples Factor Dose Log Response: VIII (U/mL) (Dose) X = Bleed ◯ = No Bleed An-53 100 2 ◯ X 63.1 1.8 X ◯ X 39.8 1.6 X An-68 63.1 1.8 ◯ ◯ 39.8 1.6 X ◯ X 25.1 1.4 X Saline 0 N/A X X hFVIII 200 2.3 ◯ ◯ Antigenicity of Ancestral FVIII

An obstacle in the treatment of hemophilia A patients with protein replacement is the development of inhibitors. In naïve hemophilia A populations, infusions of picomolar concentrations of exogenous FVIII is sufficient to generate a robust adaptive immune response. Efforts to identify and remove immunogenic epitopes within human FVIII have largely utilized a panel of well characterized mouse anti-human monoclonal antibodies (MAbs).

Using a subset of MAbs with inhibitor titers over 1000 BU/mg and specificity targeting the A2 and C2 domains, ancestral FVIII were tested as an antigenic substrate via ELISA. Despite sharing 95% identity to modern human, An-53 displays markedly reduced cross-reactivity within the A2 and C2 domains. All group A MAbs are known to bind a major epitope in the highly immunogenic 484-508 loop of human FVIII, however, only MAbs 4A4 and G32 demonstrate cross-reactivity to An-53 (FIG. 5). These MAbs were explored for inhibitor titers against An-53 and show that G32 has an inhibitory titer 45-fold less compared to human FVIII while 4A4 has an equivalent titer against both human and An-53. An-53 has 4 residue substitutions within the 484-508 loop which may explain the absence of cross reactivity and/or inhibition by these potent A2 domain inhibitors. In addition to the 484-508 epitope, homolog scanning mutagenesis of human FVIII with porcine FVIII has previously shown that a major epitope of 4A4 lies within residues 403-444, but has not been defined. Alignment of several FVIII A2 domain sequences either possessing or lacking the 4A4 epitope allows for the identification of 4 residues that may be responsible for altered binding affinity. Three of these residues, Asp403, Gln410, and Asn414 are distal to the 484-508 surface loop and therefore unlikely to contribute significant binding energy. However, Glu434 is within close proximity to Ile508. Mutation of E434 to valine as observed in porcine FVIII resulted in a >4,000 fold reduction in 4A4 inhibition to undetectable levels as measured by modified Bethesda assay (FIG. 6).

Hydrodynamic Delivery of Two an-53 Plasmid DNA (Encoding AN-53 and AN-56) in an AAV Expression Cassette

The use of AAV as a gene therapy application for hemophilia A has been explored extensively by many academic and commercial laboratories and recently advanced into clinical testing. An AAV-based product for hemophilia A has progressed beyond pre-clinical investigation. The major limitation to all gene therapy protocols for hemophilia A, both AAV and lentiviral, has continued to be the low level biosynthesis of FVIII at clinical achievable or predictably-safe vector doses. Given the enhanced biosynthesis observed for An-53 and An-56, despite high identity to human FVIII, in recombinant cell line systems, their utility was investigated in vivo following nucleic acid delivery to the liver of hemophilia A mice as a preclinical proxy for liver-directed AAV gene therapy.

An-53, An-56 and ET3 cDNAs were subcloned into an AAV expression cassette under the liver-specific HLP promoter (FIG. 7). Hemophilia A E16 mice were administered 3 μg linearized plasmid DNA (pDNA) diluted in TransIT®-EE delivery solution. A total volume equaling 10% of the mouse body weight was injected via tail vein within 4-7 seconds. Hydrodynamic injection results in DNA uptake predominantly in hepatocytes with other organs minimally affected. By placing transcriptional control under the HLP promoter, liver specificity was increase to better mimic the tropism of several AAV serotypes in clinical testing (e.g. AAV8).

Plasma FVIII levels were monitored over two weeks following injection. Peak FVIII levels were observed at day 3 for all constructs tested. Compared to the high-expression human/porcine hybrid ET3, An-53 demonstrated significantly increased and superphysiological peak FVIII levels of 2.37 units/mL. Interestingly, An-56 levels were detectable but did not exceed 0.06 units/mL throughout the experiment, suggesting that the sequence specificities allowing for enhanced biosynthesis in BHK-M cells did not convey an advantage in mouse hepatocytes. Using this system, liver codon optimized human FVIII at 3 μg per mouse was insufficient to yield detectable FVIII levels in empirical studies. The specific activity of An-53 generated in vivo was analyzed via an ELISA using cross-reactive anti-human MAbs compared to a recombinant An-53 standard curve. The FVIII produced de novo demonstrated a specific activity of 13,000±5100 U/mg.

High DNA requirement in a hydrodynamic setting can be paralleled to the viral titer required to achieve therapeutic levels of FVIII. Therefore, hydrodynamic injections were conducted using two doses, a repeat of 3 μg and 4-fold lower DNA dose at 0.75 μg per mouse. Following hydrodynamic injection, ET3 DNA was observed to display the predicted dose response. An-53 DNA produced similar FVIII activity levels at 4-fold lower DNA dose. See FIG. 8.

Human and Ancestral Chimeric Sequences

It is contemplated that ancestral and human chimeric protein can be produced. In certain embodiments, the disclosure contemplates that the human FVIII B domain deleted sequences can be replaced with Ancestral A1 and A3 sequences e.g., sp-A1 (node 68)-A2 (Human E434V mutation)-(B-partially deleted Human)-linker-an-A3 (node 68)-C1-C2 (human) SEQ ID NO: 42:

MQIALFTCFFLSLFNFCSSATRRYYLGAVELSWNYMQSDLLSVLHTDTRF LPRMPTSFPFNTSIMYKKTVFVEYMDHLFNIAKPRPPWMGLLGPTIWTEV HDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQREKEDDKVFP GESHTYVWQVLKENGPMASDPPCLTYSYLSHVDLVKDLNSGLIGALLVCK EGSLSKERTQMLHQFVLLFAVFDEGKSWHSETKDSFTQAMDSASTRAWPK MHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN HRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCP EEPQWQKKNNEEMEDYDDDLDSEMDMFTLDDDNSPFIQIRSVAKKHPKTW VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYT DV [E434V mutation] TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYS SFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRS WYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMEN PGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKN NAIEPRSFAQN [B-domain deletion] SRPPSASAPK [linker] PPVLKRHQRELSALQSEQEATDYDDSMETNEDFDIYGEDIKQGPRSFQQK TRHYFIAAVERLWDYGMSTSPHVLRNRDQSGNAPQFKKVVFQEFTDGSFS QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKE DQRQGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLE RDMHSGLIGPLLICHTNTLNPAHGRQVAVQEFALFFTIFDETKSWYFTEN VERNCKTPCNIQMEDPTLKENYRFHAINGYVMDTLPGLVMAQDQRIRWYL LSMGSNENIQSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSRAG IWRVECLIGEHLQAGMSTLFLVYSKKCQTPLGMASGHIRDFQITASGQYG QWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPI IARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVT TQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVV NSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY. 

The invention claimed is:
 1. A recombinant or chimeric FVIII protein, comprising, in an N- to C-terminal direction, an A1 domain, an A2 domain, a linker, an ap domain, an A3 domain, a C1 domain, and a C2 domain, wherein the protein has a B-domain deletion, wherein the A2 and ap domains are joined by the linker, wherein the protein has FVIII activity, and wherein: the A1 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 4; the A2 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 5; the ap domain has an amino acid sequence at least 96% identical to SEQ ID NO: 7; the A3 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 8; the C1 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 9; and the C2 domain has an amino acid sequence at least 96% identical to SEQ ID NO:
 10. 2. The protein of claim 1, wherein: the A1 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 4; the A2 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 5; the ap domain has an amino acid sequence at least 99% identical to SEQ ID NO: 7; the A3 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 8; the C1 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 9; and the C2 domain has an amino acid sequence at least 99% identical to SEQ ID NO:
 10. 3. The protein of claim 1, wherein the linker comprises an amino acid sequence between two and fifty amino acids in length.
 4. The protein of claim 1 comprising the amino acid sequence set forth as SEQ ID NO: 39 without the signal peptide of residues 1-19.
 5. A nucleic acid encoding the protein of claim
 1. 6. A pharmaceutical composition comprising the protein of claim
 1. 7. A method of inducing blood clotting comprising administering an effective amount of a pharmaceutical composition of claim 6 to a subject in need thereof.
 8. The protein of claim 1, wherein the linker comprises an amino acid sequence set forth as SFFQNPPVLKRHQR (residues 759-772 of SEQ ID NO: 39).
 9. The protein of claim 2, wherein the linker comprises an amino acid sequence set forth as SFFQNPPVLKRHQR (residues 759-772 of SEQ ID NO: 39).
 10. The protein of claim 1, comprising an amino acid sequence at least 96% identical to SEQ ID NO: 39 without the signal peptide of residues 1-19.
 11. The protein of claim 10, comprising an amino acid sequence at least 99% identical to SEQ ID NO: 39 without the signal peptide of residues 1-19.
 12. The protein of claim 3, wherein the linker comprises a PACE/furin processing sequence set forth as RHQR (SEQ ID NO: 245). 